Background
==========

Rapid and accurate detection, identification and susceptibility testing of mycobacteria remains important i) because the overall incidence of tuberculosis is increasing, also due to the HIV pandemic \[[@B1]\], ii) because of the increasing resistance to antituberculous agents \[[@B2]\] and iii) because an increasing number of mycobacterial species are being recognized as potentially pathogenic \[[@B3]\]. The importance of *M. avium* infection has increased in HIV patients \[[@B4]\], and clinical infections have been described with species like *M. heidelbergense*\[[@B5]\], *M. conspicuum*\[[@B6]\], *M. branderi* \[[@B7]\] and *M. interjectum*\[[@B8],[@B9]\], which have been recognized only recently.

Current DNA amplification based diagnostic tests are expensive, have limited senstivity, are usually restricted to the detection of *M. tuberculosis* only and provide no or limited information on susceptibility (e.g. rifampicin only: RifTB LiPA, Innogenetics, Zwijnaarde, Belgium). Therefore, the need for culture has not been circumvented. The CDC decided to restrict the use of genotypic tests to confirmation of smear positive samples, so that they cannot be used to test the large number of specimens processed for mycobacterial detection every year in an average laboratory \[[@B10]\]. Only recently, the enhanced AMTD (Gen-Probe, San Diego, CA) gained FDA approval for direct detection of *M. tuberculosis* from smear-negative samples, but several problems are reported \[e.g. \[[@B11]\], and the high costs keep restricting its use.

Here, we present our findings with the use of ARDRA \[[@B12]\] for the identification of *Mycobacterium* species. The method consists of amplification of the 16S rRNA gene (rDNA) and subsequent restriction digestion of the amplicon. The restriction patterns obtained with different restriction enzymes and combination of these patterns into a restriction profile was shown to enable identification of most clinically important mycobacteria by comparison of the obtained profiles with a library of ARDRA profiles obtained for reference strains of different species \[[@B12]\]. This PCR-RFLP analysis of the 16S rRNA gene, was published almost simultaneously with the more widely used technique (known as PRA), which is based on the amplification of the *hsp65* gene \[[@B13]-[@B15]\].

Results
=======

The initial study describing the applicability of ARDRA for the identification of mycobacteria \[[@B12]\] used universal bacterial primers. However, this sometimes resulted in the false positive amplification from decontaminated samples of organisms other than mycobacteria. Therefore primers were developed, aimed at more specific amplification of mycobacteria. During the three year evaluation period, of which the results are reported here, amplification of nonmycobacterial organisms occurred in two cases. These organisms, namely *Corynebacterium glutamicum* and *Actinomyces odontolyticum,* stained acid fast on direct smear and are relatives of the mycobacteria.

The restriction patterns obtained with the enzymes *Hha*I (isoschizomer of *CfoI*), *Mbo*I and *Rsa*I for the different species are numbered arbitrarily and are presented in Figures [1](#F1){ref-type="fig"}, [2](#F2){ref-type="fig"} and [3](#F3){ref-type="fig"} respectively. Figures [4](#F4){ref-type="fig"},[5](#F5){ref-type="fig"},[6](#F6){ref-type="fig"} represent the restriction patterns obtained when digital restriction is carried out with the same enzymes on published GenBank sequences. The combination of these patterns is designed as ARDRA profiles and these are listed in Table [1](#T1){ref-type="table"}. For example, strains of the *M. tuberculosis* complex can be recognized by an ARDRA profile 1-1-1, while *M. grodonae* strains have ARDRA profile 8-4-2. For some species the ARDRA pattern obtained with enzyme is already characteristic, e.g. *Hha*I 1 is observed only for species of the *M. tuberculosis* complex.

![*Hha*I (*Cfo*I) restriction patterns of amplified mycobacterial 16S rRNA genes. Legend: M: marker (100 base pair ladder, Fermentas, Vilnius, Lithuania)](1471-2180-2-4-1){#F1}

![*Mbo*I restriction patterns of amplified mycobacterial 16S rRNA genes. Legend: M: marker (100 base pair ladder, Fermentas, Vilnius, Lithuania)](1471-2180-2-4-2){#F2}

![*Rsa*I restriction patterns of amplified mycobacterial 16S rRNA genes. Legend: M: marker (100 base pair ladder, Fermentas, Vilnius, Lithuania)](1471-2180-2-4-3){#F3}

![*Hha*I (*Cfo*I) restriction patterns of mycobacterial 16S rRNA genes, theoretically calculated using RFLP (Applied Maths) and published GenBank sequences. Graphical representation and table of restriction fragment lengths for each of the possible patterns.](1471-2180-2-4-4){#F4}

![*Mbo*I restriction patterns of mycobacterial 16S rRNA genes, theoretically calculated using RFLP (Applied Maths) and published GenBank sequences. Graphical representation and table of restriction fragment lengths for each of the possible patterns.](1471-2180-2-4-5){#F5}

![*Rsa*I restriction patterns of mycobacterial 16S rRNA genes, theoretically calculated using RFLP (Applied Maths) and published GenBank sequences. Graphical representation and table of restriction fragment lengths for each of the possible patterns.](1471-2180-2-4-6){#F6}

::: {#T1 .table-wrap}
::: {.caption}
######

Library of ARDRA profiles (combination of restriction patterns) obtained for mycobacterial species.
:::

  **Species**                   **Genbank Number**   ***Hha*I**   ***Mbo*I**   ***Rsa*I**   ***Bst*UI**   **Reference strains used^a^**
  ----------------------------- -------------------- ------------ ------------ ------------ ------------- -------------------------------
  *M. tuberculosis* complex     X52917               1            1            1            1             ITG 8021, IPB 92/0805
  *M. conspicuum*               X88922               1\'          1            2            2
  *M. intracellulare*           X52927               1\'          2            2            3             ITG 5913, ITG 5917
  *M. gastri*                   X52919               1\'          4            1            1
  *M. kansasii*                 M29575               1\'          4            1            1             ITG 8201
  *M. bohemicum*                AJ277283             1\'          4            1            3
  *M. haemophilum*              U06638               1\'          4            1            3             ITG 3065
  *M. malmoense*                AF152560             1\'          4            1            3             ITG 940611
  *M. szulgai*                  X52926               1\'          4            2            1             ITG 4981
  *M. scrofulaceum*             X52924               1\'          4            2            3             ITG 4988
  *M. xenopi*                   X52929               1\'          4            3            2             ITG 4986
  *M. heckeshornense*           AF174290             1\'          4            3            4
  *M. marinum*                  AF251565             2            1            1            1             ITG 1728
  *M. asiaticum*                M29556               2            1            2            5             ITG 8182
  *M. terrae*                   X52925               2            1            4            2             ITG 4922
  *M. ulcerans*                 Z13990               2            1            9            1             ITG 724, ITG 1837
  *M. avium*                    AF306455             2            2            2            3             ITG 4991, ITG 2666
  *M. botniense*                AJ012756             2            4            3            2
  *M. terrae*-like MCRO6^b^     X93032               2            6            4            7
  *M. nonchromogenicum*         X52928               2            6            4            7             ITG 4980
  *M. phlei*                    M29566               3            1            6            2\'
  *M. elephantis*               AJ010747             3            1            8            3\'
  *M. chelonae* group IV^c^     AJ416940             3            3            5            2\'           ITG 7701
  *M. abscessus* group III^c^   AJ419970             3            3            6            2\'           ITG 98-1296
  *M. chelonae* group 1^c^      AJ419969             3            3            6            2\'           ITG 95-0026
  *M. immunogenum*              AJ011771             3            3            6            2\'           ITG 98-1289
  *M. farcinogenes*             AF055333             4            1            6            7
  *M. fortuitum*                X52933               4            1            6            7             SLZ A046
  *M. senegalense*              M29567               4            1            6            7
  *M. septicum*                 AF111809             4            1            6            7             ITG 4166
  *M. intermedium*              X67847               5            4            6            1\'
  *M. interjectum*              AJ272088             5            4            6            2\'           ITG 96-116
  *M. heidelbergense*           X70960               5            4            8            2\'
  *M. simiae*                   X52931               5            7            6            2\'           ITG 4485
  *M. lentiflavum*              X80769               5            7            6            2\'
  *M. peregrinum*^d^            X52921               7            1            6            9             ITG 99-2069, ATCC 14467
  *M. gordonae*                 M29563               8            4            2            5             ITG 7838
  *M. wolinskyi*                Y12871               10           1            1            2\'
  *M. species*^a^               AF028712             10           1            1            2\'
  *M. goodii*                   Y12872               10           1            2            2\'
  *M. smegmatis*                AJ131761             10           1            2            2\'           ITG 4995
  *M. kubicae*                  AF133902             10           1            6            1\'
  *M. tusciae*                  AF058299             10           1            6            2\'
  *M. flavescens*               X52932               10           1            6            2\'           VUB A016
  *M. genavense*                X60070               10           7            6            2\'           ITG 97-102
  *M. triplex*                  U57632               10           7            6            2\'
  *M. chelonae* group II^c^     AJ419968             13           3            6            6             ITG 96-0295
  *M. branderi*                 X82234               14           1            1            9
  *M. celatum*^e^               L08169               14           1            1            10
  *M. triviale*                 X88924               15           1            10           10\'
  *M. aurum*                    X55595               16           1            6            8

a\. ITG: Institute for Tropical Medicine, Antwerp, Belgium; IPB: Institute Pasteur du Brabant, Brussels, Belgium; SLZ: Streeklaboratorium Zeeland, Goes, the Netherlands; VUB: Free University of Brussels, Brussels, Belgium. b. Strains designated MCRO6 have been studied by Turenne *et al.*\[[@B22]\] and Torkko *et al.*\[[@B33]\]. c. Sequences determined in this study. Roman numbering according to Portaels *et al.*\[[@B16]\], who distinguished four groups within the *M. abscessus/M. chelonae* complex, based on the 16S--23S rRNA spacer region. d. The GenBank sequence (AF058712) of strain ATCC 14467, submitted to GenBank as *M. peregrinum,* did not cluster within the *M. fortuitum* complex (Figure [8](#F8){ref-type="fig"}) and was highly similar to sequence Y12871 (*M. wolinskyi*). Moreover, the ARDRA profile calculated from sequence AF058712 (10-1-1-2\') did not correspond with the profile we obtained for strain ATCC 14467 (4-1-6-7). The sequence obtained in this study from strain ATCC 14467 (submitted as AJ422046) was identical to the *M. fortuitum* sequence X52933. Sequence AF058712 is indicated as *M. species* in the table. e. Received as *M. xenopi.*
:::

Most mycobacterial species could be readily identified by comparison of the obtained ARDRA profile with the profiles from Table [1](#T1){ref-type="table"}. The species of the *M. tuberculosis* complex can not be differentiated on the basis of the 16S rDNA sequence, and therefore restriction digestion of this gene could not either. Most of the clinically relevant and the most abundant species were readily differentiated from each other. The following species could not be differentiated from each other after digestion with *Hha*I, *Mbo*I, *Rsa*I and *Bst*UI: *M. gastri* and *M. kansasii* (1\'-4-1-1), *M. bohemicum, M. haemophilum* and *M. malmoense* (1\'-4-1-3), MCRO6 and *M. nonchromogenicum* (2-6-4-7), *M. chelonae* group I, *M. abscessus* and *M. immunogenicum* (3-3-6-2\'), *M. farcinogenes, M. fortuitum, M. senegalense* and *M. septicum* (4-1-6-7), *M. simiae* and *M. lentiflavum* (5-7-6-2\'), *M. goodii* and *M. smegmatis* (10-1-2-2\'), *M. tusciae* and *M. flavescens* (10-1-6-2\'), and finally *M. genavense* and *M. triplex* (10-7-6-2\').

During 1998--2000, approximately 3500 samples were sent to the laboratory for direct smear examination and mycobacterial culture. Of these, 151 specimens, from 149 patients, were culture positive, and 20% of these were also smear positive. Table [2](#T2){ref-type="table"} summarizes the obtained identifications. For 148 of the 151 isolates, identification by ARDRA was straightforward and was obtained after an average of 36 hours after receipt of the cultured strain. Only three difficulties were encountered. One isolate presented with the ARDRA profile 5-4-6, not present in the ARDRA library at that time. Sequencing lead to an identification as *M. interjectum*\[[@B8]\], a species that was not yet covered by the library. A second isolate was first misidentified as *M. xenopi* (profile 1\'-4-3), but later on it was observed that the *Hha*I fingerprint differed clearly from the *M. xenopi Hha*I 1\' pattern by its low molecular size fragments (Figure [1](#F1){ref-type="fig"}). This *Hha*I pattern was designated *Hha*I 1\", resulting in the unique ARDRA profile 1\"-4-3. The isolate was identified as *M. heckeshornense* by sequencing of the 16S rRNA gene, and can now also be identified by ARDRA. The third problematic isolate had ARDRA profile 1-1-3, again a profile that had never been observed for any mycobacterial strain studied thus far. Sequencing of the 16S rRNA gave a 99.8 % similarity to the 16S rRNA sequence of the type strain of *M. tuberculosis.* The sequence revealed a mutation at *E. coli* position 646 from A to G, abolishing the *Rsa*I restriction site GTAC at that place. This mutation shifts the *Rsa*I pattern 1 to *Rsa*I pattern 3 because the two fragments of resp. 620 and 180 bp are replaced by a single fragment of 800 bp. Further morphological and biochemical tests revealed an identification as probably *M. africanum,* one of the species of the *M. tuberculosis* complex. It should be mentioned that *M. africanum* reference strains used in a previous study \[[@B12]\], were found to have the regular *M. tuberculosis* complex ARDRA profile 1-1-1.

::: {#T2 .table-wrap}
::: {.caption}
######

Distribution of the *Mycobacterium* species during 1998--2000 in the Ghent University Hospital, number of smear positives and time to positivity of culture.
:::

  *Mycobacterium* species     Number of isolates (%)   Number of samples positive on direct smear ^a^ (% of culture positive)   Average time until positive culture in days (smear positives; negatives)
  --------------------------- ------------------------ ------------------------------------------------------------------------ --------------------------------------------------------------------------
  *M. tuberculosis* complex   61 (40)                  26 (46^b^)                                                               16.7 (12.9; 20)
  *M. avium*                  12                       2                                                                        6.5
  *M. heckeshornense*         1                        1                                                                        32.5
  *M. chelonae*               4                        0                                                                        18
  *M. fortuitum*              2                        0                                                                        35.5
  *M. gordonae*               41 (27)                  1                                                                        22.3
  *M. interjectum*            1                        0                                                                        30.5
  *M. intracellulare*         1                        0                                                                        11.6
  *M. kansasii/M. gastri*     2                        0                                                                        No data
  *M. malmoense*              1                        0                                                                        No data
  *M. szulgai*                3                        0                                                                        20.3
  *M. xenopi*                 22 (15)                  0                                                                        38.1
  *Total*                     151                      40 (26)

^a^: No data for 6 of the 61 samples. All six of these samples were *M. tuberculosis* culture positive. ^b.^ Calculated as 26 positives on a total of those 56 culture positives for which data on smear positivity were available.
:::

To construct artificial ARDRA patterns for the recently described species, we applied the programme RFLP on the published Genbank sequences. The resulting, theoretically to be expected, ARDRA profiles are presented in Table [1](#T1){ref-type="table"}.

*M. tuberculosis* was found to be the most prevalent species, with 40% of the isolates, followed by *M. gordonae* and *M. xenopi,* species mostly isolated form non-pulmonar samples with low clinical relevance.

Similarity calculation of published sequences and of sequences obtained in this study (Figure [8](#F8){ref-type="fig"}) indicates that the *M. abscessus/M. chelonae* complex (*M. chehnae, M. abscessus* and *M. immunogenum*) clusters separately from all other mycobacteria. Portaels *et al.*\[[@B16]\] described the presence of four groups within the *M. abscessus/M. chelonae* complex, based on the 16S--23S spacer sequences, as used in the INNO-LiPA Mycobacteria kit (Innogenetics, Zwijnaarde, Belgium). Groups II and IV consisted of genuine *M. chelonae* strains, which were usually from environmental sources. These two groups have characteristic ARDRA profiles, namely 13-3-6 and 3-3-5. Group III, which was found to represent genuine *M. abscessus* and *M. chelonae* group I, which most probably corresponds with *M. immunogenum,* a *M. abscessus*-like species that was recently described as being frequently involved in bronchoscope related pseudo-outbreaks \[[@B17]\], can not be distinguished from each other by ARDRA (profile 3-3-6). Strains of groups I and III are usually isolated from clinical samples, with only group III strains (genuine *M. abscessus*) being pathogenic \[[@B16]\].

![*Bst*UI restriction patterns of mycobacterial 16S rRNA genes, theoretically calculated using RFLP (Applied Maths) and published GenBank sequences. Graphical representation and table of restriction fragment lengths for each of the possible patterns.](1471-2180-2-4-7){#F7}

![Neighbour-joining similarity tree for 16S rRNA gene sequences of most mycobacterial species. Legend: *N. asteroides* ATCC 49872 (Genbank Z82229) was used as the outgroup. Table [1](#T1){ref-type="table"} lists the GenBank accession numbers of the sequences used to construct this tree. ARDRA patterns for *Hha*I, *Mbo*I, *Rsa*I and *Bst*UI are listed after the species name. a: GenBank AF028712. Erroneously listed in GenBank as *M. peregrinum* (see also legend of Table [1](#T1){ref-type="table"}). b: *M. gastri* clusters below 100% with *M. kansasii,* although it is generally agreed that the 16S rRNA gene sequences for *M. kansasii* and *M. gastri* are identical. This can be explained by the fact that the only available GenBank *M. gastri* sequence (X52919) contained several ambiguities. c. *M. lentiflavum,* initially not included in the manuscript is not presented in this tree. It clusters close to the branch including *M. heidelbergense, M. simiae, M. triplex* and *M. genavense.*](1471-2180-2-4-8){#F8}

*M. tuberculosis* was the most frequently cultured species (40% of all culture positives), followed by *M. gordonae* (27%) and *M. xenopi* (15%) The latter two species were mostly isolated from non-pulmonar samples. 46% of the samples culture positive for *M. tuberculosis,* were auramine staining positive, and the average time of incubation until a positive liquid culture of *M. tuberculosis* was 12.9 days for the auramine positive samples and 20 days for auramine negative samples. The range of time to positivity for all *M. tuberculosis* positive samples was between 6 and 29 days. The average culture times for species other than *M. tuberculosis,* as far as data were available, are presented in Table [2](#T2){ref-type="table"}.

Discussion
==========

Restriction analysis of the amplified 16S rRNA gene, or amplified rDNA restriction analysis (ARDRA) was introduced into the Laboratory for Bacteriology of the Ghent University Hospital for the identification of cultured mycobacteria in 1993 \[[@B12]\]. Since then, several comparable approaches, based on restriction digestion of the amplified rRNA genes and spacer regions have been described \[[@B18]-[@B21]\]. During that period, this approach has been updated and refined. This was possible due to some technical changes, like increased quality control of gel electrophoresis and pattern interpretation and the use of primers specific for species of the order of the Actinomycetales instead of universal bacterial primers. Refinement was also possible because of the improvement of mycobacterial taxonomy and the possibility offered by PCR-RFLP techniques, like ARDRA, to easily adapt to this new information. Indeed, when new species are described, there is no need to develop new probes or primers. Instead, new ARDRA profiles can be easily added to the existing library. Also, ARDRA profiles for newly described species can be predicted by applying computer aided digestion of the available GenBank sequences, given the availability of sequences of sufficient quality \[[@B22]\].

ARDRA was found to be a useful tool for identification of mycobacterial isolates in a clinical routine laboratory, because of its speed -- compared to phenotypic identification, its reliability, practical applicability, flexibility and the possibility to identify most nontuberculous mycobacteria together with and at the same cost as *M. tuberculosis,* at an affordable price. Of the 151 isolates during the last three years, 148 could be identified without problems. The other three isolates, respectively *M. interjectum, M. heckeshornense* and an *M. africanum*-like strain, should be identifiable when met again in the future, since they presented with specific ARDRA profiles.

Practical applicability of ARDRA
--------------------------------

The theoretical turnaround time of ARDRA is 6 hours, and the average identification time in practice during this study was 36 hours. It should be emphasized that the technique was not fully implemented in the routine laboratory, but was carried out by the research laboratory technicians, which means that the practical turnaround time should be far less than 36 hours in a routine diagnostic laboratory. Technically, ARDRA is nondemanding, comprising only basic molecular biology techniques like simple DNA extraction, PCR, restriction digestion and submarine agarose gel electrophoresis.

General considerations
----------------------

We have addressed previously the several limitations of molecular biology based detection in diagnostic bacteriology \[[@B23]\]. Others agree that the expectations that DNA amplification technologies would supplant microscopy, accurately predict culture results and provide an immediate definitive diagnosis were premature and that these claims have to be replaced with a more realistic view of the limitations and of the practical value of molecular diagnostics of tuberculosis \[[@B10],[@B24],[@B25]\]. Also the expectation that susceptibility would be carried out solely by means of DNA technology, had to be moderated \[[@B26]\]. Despite the fact that during the last ten years a tremendous effort, both in academic and commercial research, has been put into the applicability of nucleotide amplification techniques for the detection of mycobacteria directly from clinical samples, the CDC approved application of these techniques only for smear positive samples. This implicates that for 52% of the culture positive samples with *M. tuberculosis* encountered in this study, DNA technology would not have accelerated detection, since microscopy was negative. Recently, AMTD2 (GenProbe) gained FDA approval for testing smear negative sputa, but the cost of the technique keeps limiting its use to only those smear negative samples with strong clinical suspicion of tuberculosis. Moreover, since this kind of direct detection amplification technology is technically demanding or requires specialized equipment and kits, many laboratories carry out these tests only at well-set time intervals \[e.g. \[[@B11]\]\], delaying diagnosis with several days on average, and as such loosing some of the time gain offered by these direct detection methods. As a final remark, one should keep in mind that direct detection without direct antimicrobial susceptibility testing does not obviate the need for culture after all.

ARDRA compared to other culture based genotypic identification techniques
-------------------------------------------------------------------------

ARDRA and other gene restriction techniques \[[@B14],[@B15],[@B21],[@B27],[@B28]\] have been developed as a practical short cut to full sequence determination. From this study and others it is clear that the discriminatory power of these RFLP approaches for identification of mycobacteria is almost as high as that of sequencing. The discriminatory power and reliability of a commercially available rRNA-spacer based hybridisation assay (INNO-LiPA Mycobacteria) is high, but again this approach is somewhat more laborious and more expensive than in house PCR-RFLP-based techniques. Restriction digestion of a 439 bp stretch of the *hsp65* gene for identification of mycobacteria was described almost simultaneously with ARDRA and designated PCR-RFLP Analysis (PRA) \[[@B15]\]. Several laboratories have published their experience using this technique \[e.g. \[[@B14],[@B27],[@B28]\]\]. Possible drawbacks of *hsp65* gene restriction analysis are the smaller sized restriction fragments and the higher intraspecific variability, which may make interpretation more difficult. The small size differences have led to the use of polyacrylamide gel electrophoresis \[[@B27]\], which is less practical than agarose gel electrophoresis and the interpretation difficulties in general have led to reconsideration of the *hsp65* gene restriction profiles used thus far \[[@B28]\]. Comparable remarks can be made for PCR-RFLP analysis of the rRNA spacer region \[[@B21]\].

Future developments
-------------------

At present, in an effort to have the best of both worlds, we are performing a double PCR, directly on smear positive, decontaminated samples, extracting DNA with the commerially available QiaAmp Tissue kit (Qiagen, Hilden, Germany). One PCR attempts to amplify the full length 16S rRNA gene (1500 bp), which can be used for ARDRA, at an annealing at 55°C, and one PCR amplifies a 123 bp region of the IS6110 at an optimal annealing of 68.6°C \[[@B29]\]. Both PCRs are carried out simultaneously in a T-gradient thermocycler (Biometra, Göttingen, Germany), programmed to have both annealing temperatures in the 96 well block. In case of the presence of the 123 bp fragment on an agarose gel, the identification of *M. tuberculosis* is completed, and can optionally be confirmed with ARDRA. Due to the higher sensitivity of the IS6110 PCR compared to the rDNA PCR, we could amplify the IS6110 fragment of *M. tuberculosis* directly from all smear positive, *M. tuberculosis* culture positive samples thus far. In case only the 16S rDNA fragment of 1500 bp is present, the absence of *M. tuberculosis* can be confirmed by ARDRA which also immediately provides with the identification of the *Mycobacterium* species other than tuberculosis. Thus far, this yielded in all cases an identification as a nontuberculous strain, confirming the absence of *M. tuberculosis.* Also in case both amplifications of the smear positive sample remain negative, this can be interpreted as the absence of *M. tuberculosis.* A positive culture is then awaited to identify the nontuberculous organism with ARDRA.

Taxonomical considerations
--------------------------

This study also confirmed the robustness of ARDRA based identifications. For example, for the first study \[[@B12]\], we received strains identified by reference laboratories as *M. avium* and *M. xenopi,* using phenotypic methods. During the first study already, some strains that had been sent as *M. avium* could be shown by ARDRA (and subsequent confirmation by another laboratory) to be *M. scrofulaceum.* For *M. xenopi,* ARDRA indicated the presence of several groups. In the meantime mycobacterial taxonomy has been refined and the groups we indicated as *M. xenopi* A and *M. xenopi* B \[[@B12]\] appear to correspond to genuine *M. xenopi,* resp. *M. celatum* (ITG 6147)\[[@B30]\], as becomes apparent when applying the programme RFLP on the 16S rRNA sequence data we obtained. Similarly, strains that we classified as *M. fortuitum* A and B \[[@B12]\], appear to correspond to *M. fortuitum* subsp. *fortuitum,* resp. *M. peregrinum.*

Materials and Methods
=====================

Strains
-------

A collection of well characterized reference strains belonging to different mycobacterial species was used to create an ARDRA profile library. The strains used are listed in Table [1](#T1){ref-type="table"}. The 151 clinical strains used in this evaluation were collected during the period between January 1998 and December 2000 in the routine clinical laboratory of the Ghent University Hospital.

Processing and culturing of the samples
---------------------------------------

Decontamination of the samples was done by mixing 1 ml of sample with 1 ml of decontamination buffer (3% NaOH/N-acetyl L-cysteine (NALC)). After 15 min of incubation at room temperature the mixture was neutralized by adding 40 ml of 0.067 M phosphate buffer (pH 6.8), followed by centrifugation at 11600 g during 15 min. The supernatant was removed and part of the pellet was used for auramine staining and microscopy. The remaining pellet was suspended in 1 ml of phosphate buffer and used for inoculation of culture media. 100 μl was used for inoculation of a solid medium (Ogawa, Sanofi-Pasteur, Marnes la Coquette, France) and 500 μl for the inoculation of a liquid medium. During this study the automated liquid culture system was changed from the Bactec system (Becton Dickinson, Cockeysville, Md.) to the 3D BacT/Alert system (Organon Teknika, Boxtel, The Netherlands).

DNA extraction
--------------

Starting from liquid culture, 500 μl of a positive culture was transferred to a 1.5 ml screw cap Eppendorf tube. After centrifugation at 13.000 rpm for 15 minutes, the supernatant was removed and the resulting pellet was resuspended in 50 μl TE buffer (100 mM Tris-HCl -- 10 mM EDTA, pH 8.0). The mixture was heated for 30 minutes at 95°C, followed by a freezing step at -20°C for at least 30 minutes. Starting from solid culture, a loopful of a bacterial colony was suspended in 500 μl of TE buffer. The mixture was heated at 95°C for 30 minutes, followed by a freezing step at -20°C for at least 30 minutes. Prior to PCR, DNA extracts were thawed at 4°C and centrifuged shortly to pellet the debris.

***ARDRA*** for mycobacteria consists of the amplification of the 16S rRNA gene, followed by separate restriction digestion with *Hha*I, *Mbo*I and *Rsa*I The combination of the three obtained fingerprints is designated an ARDRA profile which can be compared with a library of ARDRA profiles, obtained from well-identified mycobacterial strains. In some cases, more discriminatory identification is possible by additional restriction with *Bst*UI.

Amplification of the 16S rRNA gene
----------------------------------

The primers used to amplify the full length 16S rRNA gene (approximately 1500 bp) were MBUZ1 (GAC GAA CGC TGG CGG CGT GCT TAA C) and MBUZ2 (CGT CCC AAT CGC CGA TC). These primers are designated to amplify only the 16S rRNA gene for species of the order Actinomycetales. The PCR mixture consisted of 25 μl Qiagen Mastermix (Qiagen, Hilden, Germany), 0.2 μM of each primer, 5 μl of DNA extract, and was adjusted to 50 μl with distilled water. Thermal cycling consisted of an initial denaturation of 5 min at 94°C, followed by three cycles of 1 min at 94°C, 2 min at 55°C and 1 min at 72°C, followed by 30 cycles of 20 sec at 94°C, 1 min at 55°C and 1 min 72°C, with a final extension of 7 min at 72°C, and cooling at 10°C.

***Amplification of 123 bp of the IS6110 region*** was carried out as described \[[@B29]\] after DNA extraction from decontaminated sputum samples using the QiaAmp Tissue kit (QiaGen, Hilden, Germany).

Restriction digestion
---------------------

The restriction enzymes used were *Hha*I (isoschizomer of CfoI)(Amersham Pharmacia Biotech Benelux, Roosendaal, the Netherlands), *Mbo*I (Fermentas, Vilnius, Lithuania), *Rsa*I (Amersham Pharmacia). When necessary for further discrimination, digestion with *Bst*UI (New England Biolabs, Beverly, Ma.) was carried out. Each 16S rDNA amplicon was divided in three separate tubes in aliquots of 10 μl, to which 10 U of the respective restriction enzymes were added, with 2 μl of the corresponding enzyme buffer (10× concentrated, final concentration 2×) and each restriction digestion mixture was adjusted to 20 μl with distilled water and incubated during 2 hours at 37°C in a heater.

Electrophoresis
---------------

The DNA restriction fragments were electrophoresed in a 2.5% agarose electrophoresis gel, containing 2% Methaphor (FMC Bioproducts, Rockland, Me.) and 0.5% MP agarose (Roche) in the presence of ethidium bromide (50 ng/ml). The gels were photographed and the fingerprints were compared visually with the overview gels (Figures [1](#F1){ref-type="fig"}, [2](#F2){ref-type="fig"} and [3](#F3){ref-type="fig"}).

16S rDNA sequencing and comparative analysis
--------------------------------------------

A fragment of the 16S rRNA gene (corresponding to positions 10-1507 in the *Escherichia coli* numbering system) was sequenced as described previously \[[@B31]\]. Sequencing primers were MB UZ1 (GACGAACGCTGGCGGCGTGCT TAAC, *E. coli* position 27-50), MB UZ2 (CGTCCCAATCGCCGATC, 1493 -- 1476), MBP1 (CCGGCCAACTACGTGCCAGC, 502 -- 522), MBP2 (CTGGAATTCCTGGTGTAGCGG, 673 -- 693), MBP3R (GCATGTCAAACCCAGGTAAGG, 1006 -- 986) and MBP4R (CCACCTTCCTCCGAGTTGACC, 1185 -- 1165)

The 16S rDNA sequences obtained in this study are indicated in Table [1](#T1){ref-type="table"}. All steps of the comparative sequence analysis were performed by using the GeneBase software package (Applied Maths, St. Martens Latem, Belgium), as described \[[@B32]\]. First, pairwise alignment using UPGMA was carried out with a gap penalty of 100 %, a unit gap cost of 20 % and an ambiguity cost of 50 % of the mismatch cost. Subsequently, global alignment -- with *N. asteroides* ATCC 49872 (Genbank Z82229) used as the outgroup -- was carried out on the region corresponding to positions 67 through 1444 of the 16S rRNA gene of *E. coli,* with costs as above. Finally, a similarity matrix of the aligned sequences was constructed by global alignment homology calculation and a gap penalty of 20 %. The neighbour-joining method was used to construct the dendrogram based on this similarity matrix. Bootstrap values were calculated.

***Theoretical calculation of restriction patterns*** was done by means of RFLP (Applied Maths), which makes it possible to obtain restriction patterns using sequences in EMBL format, for every restriction enzyme. The programme GelCompar (Applied Maths) was then used to display the obtained fingerprints.

::: {#T3 .table-wrap}
::: {.caption}
######

Overview of the theoretical ARDRA profiles of the species that were newly described since the start of the evaluation period (1998), as obtained after computer aided digestion of the published sequences (see also Figures [4](#F4){ref-type="fig"},[5](#F5){ref-type="fig"},[6](#F6){ref-type="fig"},[7](#F7){ref-type="fig"}).
:::

  **Species**                         **Source of isolation**             **Reference**   **GenBank Accession Numbers**   ***Hha*l**   ***Mbo*l**   ***Rsa*l**   ***Bst*UI**
  ----------------------------------- ----------------------------------- --------------- ------------------------------- ------------ ------------ ------------ -------------
  *M. bohemicum*                                                          \[[@B34]\]      AJ277284, U84502                1\'          4            1            3
  *M. botniense*                      Water                               \[[@B35]\]      AF012756                        2            4            3            2
  *M. elephantis*                     Elephant                            \[[@B36]\]      AF010747                        3            1            8            3\'
  *M. goodii*                         Human wounds                        \[[@B37]\]      Y12872                          10           1            \(1\)        2\'
                                                                                Y12872^a^                       10           1            2            2\'
  *M. heckeshornense*                 Lung disease                        \[[@B38]\]      AF1174290                       1            4            3            4
  *M. kubicae*                                                            \[[@B39]\]      AF133902                        10           1            6            1\'
  *M. murale*                         Walls of childrens daycare centre   \[[@B40]\]      Unpublished
  *M. septicum*                       Catheter related bacteraemia        \[[@B41]\]      AF111809                        4            1            6            7
  *M. tuberculosis* subsp. *caprae*   Goats in Spain                      \[[@B42]\]      AJ131120                        1            1            1            1
  *M. tusciae*                                                            \[[@B43]\]      AF058299                        10           1            6            2\'
  *M. wolinskyi*                      Human wounds                        \[[@B37]\]      Y12871, Y12873                  10           1            1            2\'

a\. After resolving the ambiguity (N) present at *E. coli* position 95 in the Genbank sequence Y12872. An identical base (G) is found at this position in all mycobacteria.
:::

Acknowledgements
================

We thank Leen Rigouts for biochemical identification of some of the strains.


Background
==========

The Hedgehog (Hh) proteins are evolutionarily conserved signaling molecules that control the normal growth and patterning of diverse animals including Drosophila and humans. In flies Hh is required for multiple developmental processes such as embryonic segment patterning, eye and appendage development (for reviews see \[[@B1],[@B2]\]) In vertebrates, three Hh homologues are expressed in a tissue specific manner and are responsible for the morphogenesis of various organs such as the neural tube and the limbs, and for cartilage and male germinal cell differentiation \[[@B3],[@B4]\]. In mammals, deregulation of the Hh pathway is responsible for cancers, especially basal cell carcinoma and medulloblastoma \[[@B5]-[@B7]\]. In all cases described so far, Hh initiates and/or maintains the transcription of target genes in responsive cells. Among the targets are *patched (ptc)*, which encodes a Hh receptor protein, and genes encoding signaling molecules. In Drosophila, *decapentaplegic (dpp)*, a signal of the TGFβ class, and *wingless (wg)*, a member of the Wnt family, are transcribed in response to Hh. Genes encoding related signaling molecules such as TGFβ, FGF, and Wnt are transcriptionally induced by Hh signals in vertebrates, as are genes for a variety of transcription factors.

In Drosophila, Cubitus interruptus (Ci), a zinc finger transcription factor of the vertebrate Gli family, plays a central and complex role in the transcriptional regulation of Hh target genes. Ci acts as either transcriptional activator or repressor in a Hh-dependent manner (for reviews see \[[@B1],[@B8],[@B9]\]). In the absence of Hh signal, most of Ci is cleaved to generate a 75 kD nuclear protein (Ci75-R) consisting of the N-terminus and the zinc finger DNA binding domain of the protein. Ci75-R acts as a repressor on *hh* and *dpp* transcription \[[@B10]-[@B12]\]. Ci cleavage is proteasome dependent and requires Ci phosphorylation by the protein kinase A (PKA), the activity of the kinesin-related protein Costal2 (Cos2) and of Slimb (Slb), a F-box WD-40 protein (other members of this family are known to direct ubiquitin-mediated proteolysis of specific phospho-proteins) \[[@B13]-[@B21]\]. In the absence of Hh, Ci75-R is mainly localized into the nucleus while the uncleaved fraction of Ci (155kD, Ci-155) is retained in the cytoplasm by Cos2 and Suppressor of Fused (Su(fu)), a putative PEST motif-containing protein \[[@B14],[@B19],[@B22]-[@B25]\]. The exclusion of full-length Ci155 from the nucleus is also ensured by its constitutive export \[[@B14],[@B24]\]. Hh signaling inhibits Ci proteolysis, probably by reducing Ci phosphorylation level via the action of a phosphatase \[[@B10],[@B14],[@B18]\]. This results in the accumulation of full-length Ci-155. Hedgehog reception also relieves Ci-155 cytoplasmic retention and allows Ci-155 to be translocated into the nucleus via a basic nuclear localization sequence \[[@B14],[@B18],[@B19],[@B25]\]. Nevertheless, the persistence of its export leads to the accumulation of the vast majority of Ci-155 in the cytoplasm. Last, proper induction of Hh target genes also requires Hedgehog signaling to produce, by an unknown mechanism, an activated form of Ci called Ci-A \[[@B11],[@B23]-[@B26]\]. In the absence of Hh, Su (fu) appears to prevent Ci activation. Upon Hh reception, both the ser-thr protein kinase Fused (Fu) and Cos2 counteract Su(fu) to produce Ci-A \[[@B19],[@B23]-[@B27]\].

The molecular mechanism by which Hh signaling controls Ci remains poorly understood. Both full-length and truncated forms of Ci are detected in the cytoplasm as part of one or more large molecular weight protein complexes \[[@B10],[@B28]-[@B30]\]. The cytoplasmic complex that has been studied also includes Fu and Cos2 and Su(fu) \[[@B28]-[@B31]\]. In the absence of Hh signal, Fu-Cos2-Ci ternary complex binds microtubules, probably through Cos2 \[[@B28]-[@B30]\]. Cultured cell experiments showed that Hh signal triggers the release of the Fu-Cos2-Ci complex from the microtubules \[[@B28],[@B29]\]. Concomitantly Hh signal increases the level of phosphorylation of both Fu and Cos2 \[[@B28],[@B32]\].

Our working hypothesis is that Hh signal could control Ci fate (i.e. cleavage, subcellular localization, and/or activation) by inducing changes in the activity, composition, and/or subcellular localization of the transducing cytoplasmic complex. Since Cos2 is a putative motor protein with microtubule-binding activity, it could play a central role in this process by regulating the association of the complex with the microtubules and perhaps by directing its movement to specific locations within the cell.

In order to better understand how Hh transducing complex may function, we focused on the precise relationships among its different members. The physical association of Cos2 with Fu and Ci has been previously demonstrated using gel filtration chromatography and co-immunoprecipitation from embryo and cultured cell extracts \[[@B28]-[@B30]\]. Here we have undertaken the identification and the mapping of the molecular interactions taking place between Cos2, Fu, Su(fu), and Ci using the yeast two-hybrid method and an *in vitro* biochemical assay. Our results show that (i) Cos2, as Su(fu), interacts with both the catalytic and regulatory domains of Fu and with the N-terminal part of Ci; (ii) Ci and Fu associate with Cos2 in the neck domain, located C-terminally to the motor domain. The precise identification of the interaction region of each protein *in vitro* provide new insights into the structure and possible mechanism of action of the complex during Hh signal transduction.

Results
=======

Cos2 -- Fu interaction
----------------------

*In vitro* interaction assays were done between a GST-Cos2 fusion protein expressed in bacteria and S-methionine-radiolabeled Su(fu) or Fu produced by *in vitro* translation. The Glutathion-S-tranferase was fused to the N-terminus of the full-length Cos2 protein.

No interaction was detected between GST-Cos2 and Su(fu) (Figure [1A](#F1){ref-type="fig"}). The same negative result was obtained using GST-Su(fu) and radiolabelled Cos2 (data not shown).

![**Direct specific interaction between Cos2 and Fu.** (A). GST pull down assay: *In vitro* translated, ^35^S-methionine-labeled Fu, Fu1-305, Fu306-805 or Su(fu) before (Input, Lanes 1, 4, 7 and 10) or after incubation with equal amount of, respectively, GST (used as a control; Lanes 2, 5, 8 and 11) or GST-Cos2 (Lanes 3, 6, 9 and 12). Input equals one third of the amount used in the pull down assay. (B). Yeast two hybrid assay: Left: β-galactosidase assay, Right: leucine assay. Each dot corresponds to a yeast diploid coexpressing, respectively, various regions of Cos2 (as shown below) or Rab3 fused to the DNA binding domain of LexA (DBD LexA) (rows I to V) and respectively Fu306-805, Fu437-805, GGTIIβ fused to the transactivation domain B42 (columns 1 to 3). Both assays were performed using RFY231-pSH18-34 (Rows I to IV) or EGY189-pSH18-34 (Row V). The negative control assays with Rab3 give the same result in both strains (Row I and data not shown). In combination with Fureg, Cos538-751 leads to even higher reporter activation than does Cos538-1201. This could be due to numerous causes as differences in stability of the protein fusion, in their nuclear targeting, and/or affinity for Fureg. Cos2: heptad repeats domain (AA643-990), in black Cos2 motor domain (AA172-357) in grey.](1471-213X-2-4-1){#F1}

In contrast, Fu binds specifically to the GST-Cos2 fusion protein but not to the GST protein alone (Figure [1A](#F1){ref-type="fig"}). We tested separately Fu1-305, the catalytic domain of Fu (called Fukin) and Fu306-805, the part of Fu that is not the kinase domain, referred to as the regulatory domain (called Fureg). Both domains have the capacity to specifically interact with the GST-Cos2 fusion protein (Figure [1A](#F1){ref-type="fig"}). Similar interactions were previously observed between Su(fu) and the two domains of Fu (\[[@B31]\] and data not shown).

To confirm the Cos2-Fu interaction and to further map the interacting regions we employed the yeast two-hybrid system (Figure [1B](#F1){ref-type="fig"}). Neither full-length Cos2 nor full-length Fu and Fukin could be used in this assay due to interfering activities. Indeed, as we have shown previously, the presence (but not the activity) of the kinase domain of Fu prevents any protein interaction (\[[@B31]\] and data not shown). Using a transcription inhibition assay \[[@B33]\], we found that the full-length Cos2 fusion to the GAL4 DNA binding domain, and some partial Cos2 proteins (such as Cos1-751) that contain the motor domain, do not translocate efficiently to the nucleus (data not shown). The motor domain of Cos2 (AA 172--347) might tether the fusion protein in the cytoplasm, perhaps by binding to microtubules. We therefore analyzed partial Fu and Cos2 proteins.

In the yeast two-hybrid assay, Fureg (Fu306-805) interacts specifically with the C-terminal part of Cos2 (Cos538-1201) (Figure [1B](#F1){ref-type="fig"}). Part of this region, Cos538-751, is sufficient to interact with Fureg. Cos538-751 encodes a region of Cos2 C-terminal to the putative motor domain (See also Figure [3](#F3){ref-type="fig"}) and which overlaps 14 of the 36 heptad repeats of a predicted coiled-coil motif of Cos2. In other kinesins the coiled coil repeats mediate homodimerization and belong to a flexible stalk domain involved in the movements of the motor domain along the microtubules \[[@B34]-[@B45]\].

![**Direct specific interaction between Cos2 and Ci.** (A). GST pull-down assay: *In vitro* translated, ^35^S-methionine-labeled Ci or a truncated form of Ci (Ci1-430) before (Input, Lanes 1 and 4) or after incubation with equal amount of, respectively, GST (Lanes 2 and 5) or GST-Cos2 fusion (Lanes 3 and 6). Ci: zinc finger domain in grey, activation domain in black, the approximate cleavage position is around AA703. (B). Yeast two hybrid β-galactosidase assay between various regions of Cos-2 or Rab3 in fusion with the DBD lex-A (Rows I to V) and respectively GGTIIβ, Ci, Ci1-346 or Ci340-445 in fusion with B42 (Columns 1 to 4). See also legends to Figure [1B](#F1){ref-type="fig"} and [2A](#F2){ref-type="fig"}.](1471-213X-2-4-3){#F2}

We also mapped the region of Fu that interacts with Cos2 (Figures [1B](#F1){ref-type="fig"} and [3](#F3){ref-type="fig"}). The C-terminal-most region of Fu (AA 437 to 805) is sufficient to interact with both Cos538-1201 and Cos538-751. This region of Fureg is adjacent but non-overlapping to the part of Fu (AA 306 to 436) that interacts with Su(fu) \[[@B31]\]. No interaction between Fu and the different Cos2 constructs tested was detected with smaller pieces of Fureg (Fureg306-436, Fureg437-581, Fureg306-581, Fureg582-805; data not shown).

In summary, Cos2 interacts with both the kinase and regulatory domains of Fu, just as Su(fu) does \[[@B31]\]. The regulatory domain of Fu interacts with a central region of Cos2 located C-terminally to the motor domain.

Cos2 -- Ci interaction
----------------------

*In vitro* assays between the GST-Cos2 fusion protein and radiolabeled Ci revealed a direct specific interaction between these proteins (Figure [2A](#F2){ref-type="fig"}). Again, as with Fu, the yeast two-hybrid assay did not allow the detection of an interaction between full-length Cos2 or Cos1-751 and Ci. Nevertheless, interactions consistent with this GST \"pull down\" result were detected in yeast two-hybrid assays using full-length Ci and the region of Cos2 spanning residues 348 to 546 (Cos348-546) (Figure [2B](#F2){ref-type="fig"}). This part of Cos2 is adjacent to the Fureg binding site, in a region located immediately C-terminal to the motor domain at the base of the neck region. This region has been shown to regulate the directionality of the movement of kinesins along microtubules \[[@B34]-[@B45]\].

![**Summary of the interaction domains** The interactions between Cos2, Ci, Fu, and Fureg described above and in former work \[[@B31]\] are summarized. The interaction between the region 941--1065 of Ci and Cos2 was previously shown by Wang et al. 2000 \[[@B23]\]. The domains of interactions are indicated in lines above or under each protein structure. No interaction could be detected between Cos2 and Sufu (data not shown). See also legends for Figures [1](#F1){ref-type="fig"} and [2](#F2){ref-type="fig"}. M: Cos2 motor domain, HR: Cos2 heptad repeats, DBD:Ci DNA binding domain; NLS: Ci nuclear localisation signal, AD: Ci activation domain.](1471-213X-2-4-2){#F3}

To identify the region of Ci that interacts with Cos2, partial deletions of Ci were generated and tested for interaction with Cos2 using a combination of *in vitro* and two-hybrid assays. The first 430 amino acids of Ci are sufficient to interact *in vitro* with GST-Cos2 (Figure [2A](#F2){ref-type="fig"}). In contrast the first 346 residues of Ci, which have been previously shown to bind Su (fu) \[[@B31]\], do not interact in yeast with Cos538-1201 nor with Cos348-546 constructs (data not shown). These results suggest that Cos2 interacts with a part of Ci between amino acids 346 and 430 (Figure [2B](#F2){ref-type="fig"}). Indeed, Ci340-445 is sufficient to interact in yeast with Cos348-546 (Figure [2B](#F2){ref-type="fig"}).

Thus, like Su(fu), Cos2 interacts within the N-terminal region of Ci, which is present in both Ci155 and Ci75-R. The region of Cos2 involved in this interaction is located at the base of its neck, which plays an important role in the activity of conventional kinesin.

Discussion
==========

Cos2, Fu and Su(fu) play multiple and complex roles in Ci control. Cos2 is central to this control both as a negative regulator in the absence of Hh and a positive regulator in the presence of Hh. In the absence of Hh signal, Cos2 action prevents Ci target activation by favoring Ci cleavage and by cytoplasmic retention of full-length Ci \[[@B14],[@B18],[@B23],[@B46]\]. In response to Hh, Cos2 is also required for complete Ci activation in the wing imaginal disc \[[@B19],[@B24]\]. Fu is also involved in positive and negative aspects of Ci regulation. Its kinase activity is required for Ci cytoplasmic release and activation in response to Hh signal. Its C-terminal regulatory domain, although also required for Fu kinase activity, cooperates with Cos2 and Su(fu) to negatively regulate Ci in the absence of Hh signal (see bellow) \[[@B19],[@B22],[@B25]-[@B27],[@B47]\]. Although *Su(fu)* mutants display only a very subtle phenotype, Su(fu) acts negatively in the Hh pathway. A decrease in *Su(fu)* (or cos2) dosage suppresses the effects of the loss of Fu kinase activity. *Su(fu)* loss of function aggravates cos2 phenotypes \[[@B47]\], so Su(fu) activity normally assists Cos2 in its negative regulation of Ci. Su(fu) seems to participate both in the retention of Ci in the cytoplasm and in preventing activation of full-length Ci \[[@B19],[@B26],[@B27]\].

Here, we show that Cos2 interacts directly with both Fu and Ci, as does Su(fu). This confirms and extends previous results, which showed these proteins to co-immunoprecipitate from embryo and cultured cell extracts and to be members of one or several large molecular weight cytosolic complex (es) \[[@B10],[@B28]-[@B30]\]. The mapping of the regions of each protein sufficient for interaction with its partner, *in vitro* and in yeast (Figure [3](#F3){ref-type="fig"}), sheds new light on the respective role of Fu, Su(fu), and Cos2 and on their relationships in the Hh pathway.

Our results demonstrate that Cos2 can bind to the N-terminus of Ci, in a region (amino acids 340--445) located between a region interacting with Su(fu) (amino acids 240--346) and the zinc finger domain (amino acids 453--603). Wang *et al*. also used the two-hybrid method to look for interactions between Ci and Cos2. In contrast to our results, they found no interaction of Cos2 with the N terminus of Ci \[[@B23]\]. This discrepancy can have multiple causes as the fact that their constructs lack amino acids 441 to 445 of Ci or differences in the fusion proteins tested (i.e. differences in protein folding, stability, nuclear targeting etc) or in the reporter used\... Nevertheless, they also identified a second region of interaction with Cos2, located in the C-terminus of Ci (Ci 941--1065) \[[@B23]\]. Both the Cos2/Ci941-1065 interaction and the previously reported Su(fu)/Ci240-346 interaction have been shown to be retain Ci in the cytoplasm in the absence of Hh signal \[[@B19],[@B23]\]. Strikingly, neither Su(fu) nor Cos2 interaction regions of Ci map to the amino acids 703 to 850 (Ci-cyt) region identified by Aza-Blanc *et al*. as involved in Ci cytoplasmic tethering \[[@B10]\]. One hypothesis is that the region 703--850 of Ci contains an export signal \[[@B14],[@B24]\].

The interaction of Su(fu) and Cos2 with the N-terminal part of Ci is in agreement with the presence of both full-length Ci-155 and Ci75-R in the large complex(es). Since Fu, Su(fu) and Cos2 can also be found in complex(es) of similar size \[[@B10]\], Cos2 and Su(fu) may control the activity of Ci75-R *in vivo* by a so far unknown mechanism. Several roles can be proposed for the interaction between Cos2 and the Ci N-terminus. The association could reinforce the tethering of Ci in the cytoplasm by Cos2 and Su(fu). An attractive alternative is that the association could underlie the requirement for Cos2 to activate Ci155 in response to Hh. For example, Cos2 and Su(fu) could compete to bind Ci N-terminus and Hh signaling could favor the transient binding of microtubule-unbound Cos2, leading to Su(fu) release.

We find that Cos2, like Su(fu), is able to interact with two different domains of Fu: the Fu kinase and regulatory domains. No phosporylation of Cos2 nor Su(fu) by Fu has been reported so far. In S2 cells, it has been reported that Hh induces Cos2 hyperphosphorylation in a Fu independent manner. Nevertheless, the interactions of respectively Su(fu) and Cos2 with the kinase domain of Fu suggest their direct phosphorylation by Fu. Some kinesins are regulated by phosphorylation, and Fu kinase activity could control Cos2 activity \[[@B48],[@B49]\]. The interaction between the regulatory domain of Fu and Cos2 is in agreement with the lack of Fu-Cos2 co-immunoprecipitation in *fu* mutants that are missing part or all of the regulatory domain (*fu*^*class\ II*^ mutant, see below) \[[@B28]\]. Possibly, the *in vivo* interaction of the kinase domain of Fu with Cos2 requires the binding of Cos2 to Fureg, or the interaction could be too transient to be detected in *fu*^*class\ II*^ mutants.

The interaction of the regulatory domain of Fu with Su(fu) and Cos2 provides a support for the complex genetic relationships previously described to occur between *Fu*, *Su(fu)*, and *Cos2*. Mutations in the catalytic (class I) and the regulatory (class II) domains of Fu are genetically distinguishable from each other in a *Su(fu)* or *cos2* mutant background, suggesting distinct roles for the two domains of Fu \[[@B22],[@B47],[@B50]\]. Thus, *Su(fu)* ; *fu*^*class\ I*^ double mutants are almost wild type, while *fu*^*class\ II*^; *Su(fu)* double mutants have a cos2-like phenotype associated with the activation of Ci target genes. Similarly, *fu*^*class\ II*^ alleles strongly enhance the effect of a heterozygous *cos2* mutation, though alleles *fu*^*class\ I*^ do not. These observations strongly suggest that the regulatory domain of Fu also plays a negative role in Hh signal transduction, this effect being detected only in association with *Su(fu)* or *cos2* mutations. We propose that, in the absence of Hh signal, Fureg could act as a scaffold to anchor Su(fu) and Cos2 in the complex, favoring their negative effect on Ci. In *fu*^*class\ II*^ mutants, the negative effect of Su(fu) and Cos2 would be lessened due to their weaker anchoring in the complex. This would only be revealed in the presence of a decrease in Su(fu) or Cos2 dosage that would cause a further destabilization of the complex.

In conventional kinesins, the C-terminal globular tail domain associates with vesicles. Our results show that the Cos2 C-terminus is not required to bind either to the Ci N-terminus (Ci340-445) or the Fureg domain. Instead, both interactions involve adjacent sequences located between the motor domain and the stalk homodimerisation domain. In kinesin itself, this region is called the \"neck\" domain and is important for efficient motor function. In at least some kinesin molecules the neck is involved in the determination of the direction of kinesin movement along the microtubule, in velocity, in processivity, and in mechanochemical coupling \[[@B34]-[@B45]\]. Thus, although no Cos2 motor activity has been described so far, Fu and/or Ci binding to its central region could induce structural changes and therefore modulate Cos2 motor activity. This hypothesis opens the possibility of a feedback control of Ci on Cos2 activity.

Conclusion
==========

The present data, together with published results, suggest that the existence of direct multiple interactions between Cos2, Su(fu), Fu, and Ci, each protein interacting with at least two other partners. We propose a model based upon a complex that includes Fu, Cos2, Su(fu), and Ci (Figure [4](#F4){ref-type="fig"}). Changes in the composition, activity and/or subcellular localization of this complex will control Ci fate in the absence and in the presence of Hh. In the absence of Hh signal, Su(fu) and Cos2 prevent Ci activation, Ci processing into the repressor form is favored and full-length Ci is exported from the nucleus. Upon Hh reception, Fu is activated, opposing Su(fu) to allow Ci to become an activator. Simultaneously the negative effect of Cos2 is alleviated, perhaps by release from the microtubules and/or a change in subcellular location. This lowers the rate of proteolysis of Ci into its repressor form and triggers nuclear import of full-length Ci. Furthermore, Cos2 and Fu promote the conversion of Ci 155 into an activator form, ultimately resulting in the transcription of Hh target genes.

![**Model of Hedgehog signaling** In the absence of Hh signal, Cos2 and Su(fu) binding to Ci prevents Ci activation and retain it in the cytoplasm. Most of Ci is available for cleavage in a process which is dependent upon its phosphorylation by the PKA and which involves Cos2 and Slimb. Uncleaved, full-length Ci, is actively exported from the nucleus. Upon Hh reception, Fused is activated and acts on Cos2 and Sufu, alleviating thus their negative effect on Ci. As a result, Ci cleavage is reduced, Ci155 nuclear import overcomes its export and Ci is activated. Ci activation requires Cos2 and Fu to antagonize Su(fu) negative effect. Activated nuclear Ci interact with the CBP to fully activate the transcription of Hh target genes.](1471-213X-2-4-4){#F4}

So far, each protein of the complex can serve as a direct link between two other proteins, and could in this way form a scaffold for the complex. It is important to note that the interactions observed *in vitro* may not define a singular protein complex *in vivo*. A subset of the proteins could associate at any given time, or more than one type of complex may exist simultaneously. Additional interactions, may of course contribute to the stability, organization, or activity of the complex(es). Structural data and new functional tests will be required to learn more about the multiple interactions among the components, and their multiple roles in the control of Hh signaling.

Materials and Methods
=====================

Strains
-------

RFY231: *Matα ura3-1 his3 trp1 Δ :: hisG 3lexAop-LEU2 : :leu2* was built by R. Finley (unpublished data). EGY189 *Matα ura3-1 his3 trpl-1 1lexAop-LEU2: :leu2* has a less sensitive *leu2* reporter gene than RFY231 due to a lower number of copies of *lexAop*\[[@B51]\]), RFY206: *Mata his3Δ200 leu2-3 lys2 Δ 201 ura3-52 trp1ΔhisG.* Yeast cultures and transformations were performed as in \[[@B31]\].

Plasmids
--------

The two-hybrid pEG and pJG expression vectors were derived from pEG202 (translational fusion with the DNA binding domain of lexA) and pJG4-5 (translational fusion with the activation domain B42) \[[@B33],[@B52]\]. Two numbers added after the protein name indicate the first and last residues of truncated proteins. pJGCi (full-length), pJGCi1-346 and pJGFu306-805 (pJGFureg), were described in \[[@B31]\]. pEGCos2 (full-length), pEGCos1-751, pEGCos348-546, pEGCos538-1201, pEGCos538-751, pJGCi340-445, pJGFu437-805 were built by in frame cloning of restriction fragments or PCR products. pSH18-34 carries the lacZ reporter gene \[[@B52]\]. The GST-Cos2 translational fusion was made by introducing the entire Cos2 coding sequence in frame with the glutathione S-transferase (GST) gene in pGEX-4T3. All PCR products and junctions were sequenced.

GST fusion protein binding assays
---------------------------------

GST-Cos2 and GST protein production and purification, *in vitro* coupled transcription-translation, and *in vitro* interaction assays were performed as described in \[[@B31]\]. In all the figures, the amount of radiolabelled protein used in each \"input\" lane equals one-third the amount of protein used in the assay and equivalence in the amount of GST and GST-Cos2 was checked by Coomassie blue staining. All assays were done at least twice.

Two hybrid assays
-----------------

RFY231-pSH18-34 or EGY189-pSH18-34 were transformed with pEG (lexA fusion) constructs and subsequently mated with RFY206 transformants containing the pJG constructs (B42 fusion). Geranyl-geranyl transferase II β (GGTIIβ) and Rab3 are two proteins known to interact together \[[@B53]\]. They are respectively used as negative controls in tests of the pEGCos2 constructs and the pJGCi or pJGFu constructs. Diploids were selected on SCglu-UHW. For each diploid strain, 3 μl of cell suspension were spotted on SCgal-UHW (for β-galactosidase assays) or SCgal-UHWL solid media (for leucine auxotrophy assays). Growth on SCgal-UHWL was observed after 24 hours of incubation at 30°C. β-galactosidase assays were performed by the overlay method as described in \[[@B54]\]. All assays were done at least three times.

Acknowledgements
================

We thank Russ Finley for plasmids and advice, M. Barre, F. Besse, F. Dussillol, S. Fouix, S. Szuplewski, D. Busson, H. Tricoire and the other members of both laboratories for their help in this work. We are deeply grateful to C. Lamour-Isnard for all her support and numerous stimulating discussions. This work was supported by grants from the ARC (N° 5523), by NSF-CNRS funds, and by the Howard Hughes Medical Institute.


Background
==========

Acute Intermittent Porphyria (AIP) is an autosomal dominant inherited disorder because of a partial defect in Porphobilinogen Deaminase (PBGD), the third enzyme in the heme biosynthetic pathway. As a result, the porphyrins precursors, δ-aminolevulinic acid (ALA) and Porphobilinogen (PBG) can accumulate \[[@B1]\]. Furthermore, the free heme pool would decrease and adversely affect P-450 and several important antioxidant enzymes, leading to impaired hepatic detoxification reactions \[[@B2]\]. Clinically, the disease is characterized by occasional acute attacks of abdominal pain and various neuropsychiatric symptoms. Several therapeutic drugs, anaesthetics and infections are considered the most frequent precipitating factors of acute attacks in the Argentinean population \[[@B3]\]. Specific treatments for acute attacks include high glucose intake \[[@B4]\], hemin \[[@B5]\] and folic acid in combination with glucose \[[@B6]\].

In Northern Sweden, Lithner and Wetterberg \[[@B7]\] and Hardell *et al.*\[[@B8]\] have found a clear association between AIP and hepatocellular carcinoma, also confirmed by other authors \[[@B9]\].

Moreover, ALA has been demonstrated to produce reactive oxygen species through metal-catalyzed aerobic oxidation and to cause oxidative damage to proteins, lipids and subcellular structures. Exposure of plasmid pBR322 DNA to ALA produced DNA single-strand breaks \[[@B10]\].

In this study, we have examined the effect of ALA on human hepatocarcinoma HEP G2 and HEP 3B cell lines viability. Tumor cells were exposed to different ALA concentrations and ALA-S activity repressed conditions, produced by hemin or glucose addition to the medium. DNA fragmentation was also analyzed. Northern blot analysis for p53 and Western blot analysis for CDK2 and CDK4 were evaluated to determine cells sensitivity to ALA.

Materials and methods
=====================

Chemicals
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Hemin, ALA, 3-\[4,5-dimethylthiazol-2-yl\]-2,5-diphenyltetrazolium bromide and L-glutamine were obtained from Sigma Chemical Co. (St. Louis, MO). D-Glucose, PBS, Dulbeco\'s Modified Eagles Medium (DMEM) and antibiotics were obtained from GIBCO BRL.

Cell lines and cell culture
---------------------------

HEP G2 and HEP 3B cell lines (American Type Culture Collection, Rockville, MD) were derived from a human hepatocellular carcinoma \[[@B11]-[@B13]\]. HEP G2 contained wild-type p53 \[[@B14]\] and HEP 3B cells contained deleted p53. Cell lines were grown in total darkness, at 37°C in 5% CO~2~ atmosphere in Dulbecco\'s Modified Eagle Medium supplemented with 10% fetal bovine serum, 10,000 U penicillin, 10,000 μg streptomycin and 2 mM L-glutamine.

MTT assay
---------

After exposure to drugs, the viability was determined by the MTT assay following published procedures \[[@B15]\]. Briefly, 40,000--50,000 cells in 500 μl of medium were plated onto each well of 24-well tissue culture plate. After 24 h of culture, ALA (0.5--5 mM), Hemin (8--24 μg/ml) or D-glucose (2--3 mg/ml) dissolved in PBS immediately prior to use, were added to the medium. Cells were cultured for 24 h. After exposure, the medium was aspirated and replaced with 300 μl of fresh drug-free medium containing 0.5 mg/ml (3-\[4,5-dimethylthiazol-2-yl\]-2,5-diphenyltetra-zolium bromide) MTT. The cells were incubated for an additional 4 h. This medium containing MTT was removed by inverting the plate and 200 μl of dimethylsulphoxide (DMSO) were then added to each well. After 10 minutes of shaking, absorbance in each well was read in a microplate reader at 570 nm. All experiments included untreated control cells, they were repeated three times and each concentration was tested in sextuplicate. Viability was expressed in terms of population growth, as percent of an untreated control population with standard error of the mean (SEM).

Analysis of DNA fragmentation
-----------------------------

Treated cells were grown in 75 cm^3^ flasks. Both attached and unattached cells were harvested, washed with PBS, resuspended in TNE solution (10 mM Tris-HCI, pH 7.6; 140 mM sodium chloride; 1 mM EDTA), and lysed at 37°C in 2 ml of extraction buffer (10 mM Tris-HCI, pH8.0; 100 mM EDTA, pH 8.0; 20 μg/ml pancreatic RNase; 0.5% SDS). After 2 h, proteinase K was added at a final concentration of 100 μg/ml and the mixture was incubated for another 3 h at 50°C. The DNA was extracted twice with equal volumes of phenol and once with chloroform. The DNA was then precipitated with 0.1 vol of sodium acetate (pH 4.8) and 2.5 vol of ethanol at -20°C overnight and pelleted at 13,000 g for 30 min. Samples were electrophoresed in 2% (w/v) agarose gel and DNA was visualized by ethidium bromide staining.

Northern blot analysis
----------------------

Total RNA (40 μg), from exponentially growing cells treated with different concentrations of ALA (0.5--5 mM) during 24 h, was prepared by Chomsinsky and Sacchi \[[@B16]\] method, size-fractionated on 1% agarose/5% formaldehyde gels, transferred onto nylon membrane Hybond-N+ (Amersham Pharmacia Biotech), and ultraviolet cross-linked. The blots were then prehybridized and hybridized with pTRI-p53-Human or pTRI-GAPDH-Mouse (Ambion) to 65°C with Ultrahyb solution (Ambion) and washed as manufacturer described. Blots were exposed to AGFA films at -70°C and developed. cDNA probes were labeled with \[α-^32^P\]ATP (3,000 Ci/mmol) using Kleanow (Biolabs) and random primers, for 2 hours at 37°C. Intensity of bands were analysed with ImageMaster (Amersham Pharmacia Biotech).

Western blot analysis
---------------------

Exponentially growing cells (5 × 10^6^/25 ml) were treated with different concentrations of ALA (0.5--5 mM). After 24 h of incubation, cells were washed with PBS and lysed in 500 μl of RIPA buffer (50 mM Tris pH 7.4; 150 mM NaCI; 20 mM EDTA pH 8; 0.1% SDS). After sonication (3 × 15 s) and 30 min incubation with PMSF (10 mg/ml) at 4°C, cell lysates were centrifuged at 10,000 g for 10 min at 4°C. Protein content of supernatants was determined by Bradford protein assay. Cell lysates containing equal amounts of protein (50 μg) were resolved on 12 % SDS-polyacrylamide gel electrophoresis (PAGE). Proteins were transferred onto nitrocellulose membranes Hybond ECL (Amersham Pharmacia Biotech) at 300 mA for 1 hour at 4°C using Towbin buffer (25 mM Tris; 192 mM glycine; 3.5 mM SDS; 10% Methanol; pH 8.3). Membranes were blocked by incubation in TBS (20 mM Tris; 137 mM NaCl; 0.05 % Tween 20; pH 7.6) containing 5% dry low fat milk for 1 hour at room temperature. Blots were probed with either anti-CDK2 (1:300), anti-CDK4 (1:300) or anti-actin (1:100) goat polyclonal antibody (Santa Cruz Biotech, Santa Cruz, CA). Immune complexes were detected using donkey anti goat secondary antibody (1:1,500) (Santa Cruz Biotech, Santa Cruz, CA) and were visualized using ECL reagents (Amersham Pharmacia). Intensity of bands was analyzed with Image Master (Amersham Pharmacia Biotech).

Results
=======

Cytotoxic effects of ALA in Human Cancer Cells
----------------------------------------------

Human hepatocarcinoma cells were treated with various concentrations (0.5--5 mM) of ALA and the viability of cells was determined (Figure [1](#F1){ref-type="fig"}). ALA was toxic to both hepatocarcinoma cell lines. In HEP G2 cells, cytotoxicity occurred after exposure to 0.5 mM ALA for 24 h (growth inhibition: 30%) and the highest growth inhibition (70%) was observed with the highest ALA concentration (5 mM). HEP 3B cells were more resistant to ALA than HEP G2 cells, exhibiting only 12% growth inhibition after exposure to 0.5 mM ALA for 24 h and a 40% growth inhibition with 5 mM ALA. The strange shape of the viability curves obtained in both cases, with a restoration of control viability with 2 mM ALA, cannot be explained.

![**Cytotoxic effects of ALA.** Human hepatocarcinoma cell lines (HEP G2 (circles) HEP 3B (squares)) were grown in the absence or in the presence of increasing ALA concentrations (0.5 to 5 mM) and the viability of cells was determined by the MTT assay. Viability was expressed in terms of population growth, as percent of an untreated control population with standard error of the mean (SEM).](1471-2407-2-6-1){#F1}

Hemin and D-Glucose effect
--------------------------

Simultaneous treatment of the HEP G2 cells with ALA (0.5 mM) and non-toxic concentrations of hemin (8--24 μg/ml) for 24 h resulted in a significant decrease in sensitivity to ALA (Figure [2](#F2){ref-type="fig"}), reverting its cytotoxic effect. It is worth noting that treatment of HEP G2 cells with hemin alone stimulated cell survival (20% over control). However, the same treatment of HEP 3B (ALA and hemin) increased ALA toxicity.

![**Hemin effect on ALA toxicity.** The effect of Hemin was determined by exposing HEP G2 and HEP 3B cells to 0.5 mM ALA without (white) or with 16 μg/ml Hemin (grey) for 24 h before the MTT assay was performed. Controls were grown with 16 μg/ml Hemin alone (black). Viability was expressed in terms of population growth, as percent of an untreated control population with standard error of the mean (SEM).](1471-2407-2-6-2){#F2}

Alike hemin, simultaneous treatment of the HEP G2 cells with ALA (0.5 mM) and D-glucose (2--3 mg/ml) for 24 h resulted in about two-fold decrease in sensitivity to ALA (Figure [3](#F3){ref-type="fig"}). And again the same treatment of HEP 3B (ALA and D-glucose) enhanced ALA cytotoxicity.

![**Glucose effect on ALA toxicity.** The effect of Glucose was determined by exposing HEP G2 and HEP 3B cells to 0.5 mM ALA without (white) or with 2 mg/ml glucose (grey) for 24 h before the MTT assay was performed. Controls were grown with 2 mg/ml glucose alone (black). Viability was expressed in terms of population growth, as percent of an untreated control population with standard error of the mean (SEM).](1471-2407-2-6-3){#F3}

DNA fragmentation after ALA treatment
-------------------------------------

As shown in Figure [4a](#F4){ref-type="fig"}, after 24 h exposure of HEP G2 cells to ALA (1--5 mM) both attached and unattached cells were harvested. In each case, nucleosomal DNA ladders, typical of apoptosis, were visible on agarose gel after staining with ethidium bromide. When HEP 3B cells were treated under the same conditions, nucleosomal DNA ladders were much less visualized (Figure [4b](#F4){ref-type="fig"}). Apoptosis was also corroborated morphologically by phase contrast microscopy in both cell lines. The decrease in viability was due to cytotoxicity as observed under phase contrast microscopy. ALA caused hepatoma cells first to shrink and then gradually to become dettached, starting about 14 hours after treatment as observed by phase contrast microscopy (data not shown).

![**DNA fragmentation analysis in human hepatocarcinoma cells undergoing ALA-induced apoptosis.** HEP G2 (a) or HEP 3B (b) cells were treated with ALA (0; 1; 2 or 5 mM) and DNA fragmentation was examined 24 h later. HEP 3B cells were also treated with Etoposide (Eto, 5 μM), used as positive control.](1471-2407-2-6-4){#F4}

Modulation of p53 and cell cycle in HEP G2 cells
------------------------------------------------

To elucidate the mechanisms of ALA causing apoptosis selectively, we have examined the effect of ALA on p53 mRNA levels. No any change in mRNA p53 levels for 24 h was observed in HEP G2 cells (Figure [5](#F5){ref-type="fig"}). Only at concentrations as high as 5 mM ALA, p53 mRNA expression was 25% diminished.

![**p53 mRNA expression in HEP G2 cells under ALA treatment.** Total RNA was isolated after treatment of cells with ALA (0--5 mM) during 24 h and hybridized with cDNA p53 specific probe and normalized relative to GAPDH. Intensity of bands were analysed with ImageMaster.](1471-2407-2-6-5){#F5}

When CDK2 and CDK4 protein levels were analyzed in HEP G2 cells after exposure to ALA for 24 h (Figure [6](#F6){ref-type="fig"}), both kinases decreased with increased ALA concentrations, up to 2 mM. At concentrations above 2 mM protein content increased reaching basal levels at 5 mM (Figure [6](#F6){ref-type="fig"}).

![**Western blot analysis of CDK4 and CDK2.** Proteins were isolated after treatment of cells with ALA (0--5 mM) during 24 h. Proteins were normalized to 50 μg per lane and the amount of CDK2 and CDK4 was detected using polyclonal antibodies and visualized using ECL reagents. Intensity of bands was analysed with ImageMaster.](1471-2407-2-6-6){#F6}

Discussion
==========

The heme precursor ALA accumulates under pathological conditions, such as AIP, tyrosinosis and lead poisoning, diseases often linked with increased liver cancer incidence \[[@B17]\]. The reactive oxygen species generated upon ALA metal-catalyzed oxidation, promote the formation of several radical-induced base degradation products in isolated and cellular DNA \[[@B17]\]. ALA is the precursor of Protoporphyrin IX (Proto IX). Exogenous ALA bypasses the feedback control and may induce Proto IX accumulation. Since heme-containing enzymes are essential for energy metabolism, every nucleated cell should have at least a minimal capacity to synthesize Proto IX and this is the rational for the use of Photodynamic Therapy \[[@B18]\].

ALA proved to be cytotoxic in neuroblastoma cells. This cytotoxic effect of ALA could be diminished by heme, presumably through modulation of ALA synthesis \[[@B19]\].

Here we have demonstrated the cytotoxic effect of ALA on two human hepatocellular carcinoma cell lines HEP G2 and HEP 3B, which was greater on the former. ALA effect would be attributed to its accumulation in HEP G2 cells, as it was shown by its reduction after simultaneous exposure with hemin or glucose, well-known repressors of ALA-S activity. There is evidence that following the exposure to external ALA, there is induction of endogenous ALA synthesis, and that ALA uptake is higher in more rapidly proliferating cells \[[@B20]\]. The strange shape of the viability curves is difficult to explain within the context of these experiments and could probably be due to different ALA and/or PPIX accumulation, which will be measured in future assays.

In HEP G2 cells, protection by hemin would be due to induction of the HO-1 gene, and increased formation of bilirubine, through heme degradation with its known free radicals scavenger effect \[[@B21],[@B22]\]. Glucose protection could be attributed to overcoming reduced glucose uptake provoked by ALA. ALA was reported to affect cultured chick embryo neuronal and glial cells by decreasing glucose uptake \[[@B23]\].

In HEP 3B cells, treatment with ALA and hemin or glucose produced increased cytotoxicity, indicating that the mechanism of ALA action involved would certainly be different.

Apoptosis is a physiological event occurring in response to multiple stimuli including growth factor withdrawal, radiation therapy and presence of chemotherapeutic agents. Several mechanisms regulate the apoptotic process, such as induction of a p53-dependent-pathway after DNA-damaging agents, modulation by the bcl2 family of proteins, and activation of some effectors including the interleukin-converting enzyme (ICE) family of proteases and endonucleases \[[@B24],[@B25]\].

Interestingly, ALA produced apoptosis visualized by DNA ladders, but its action did not appear to correlate with the p53 status; while HEP 3B p53 null cells are relatively resistant to ALA-induced apoptosis, HEP G2 p53 wild type cells are very sensitive to ALA-induced apoptosis. However, the treatment did not alter mRNA p53 levels as revealed by Northern blot analysis suggesting that apoptosis would be occurring through other mechanism, p53 independent.

CDKs have been implicated as modulators of apoptosis \[[@B26]\]. Some important influences that may have an impact on susceptibility to apoptosis include altered generation of reactive oxygen or nitric oxide intermediates or propensity for mitochondrial damage or protease activation.

The mammalian cell cycle is controlled by sequential activation and inactivation of the CDKs family, which are activated by cyclins and inhibited by CKIs. The regulators that operate in the different phases of the cell cycle are various.

In our experiments, CDK2 and CDK4 protein levels decreased with ALA treatment preventing cells proliferation. Considering that both kinases are involved in cell cycle progression, diminution of their levels would induce G1 arrest and lead to the onset of the apoptotic process. When ALA was added to the medium at supraphysiological concentrations (higher than 3 mM), the rate of death cells were so high that the measured kinases levels would only correspond to a minimal proportion of viable cells. Perhaps, these cells represent a part of the population in S phase and therefore, with high levels of the kinases necessary to duplicate. These results prompted us to speculate that ALA toxic effect would be effective only in those cells which would be able to enter in arrest and would not exert any effect on those which are cycling. Further studies are in progress to further support our hypothesis. Disruption of the regulatory system controlling G1 phase progression is a common event in human hepatocarcinogenesis. Overcoming alterations that have occurred in the G1 phase regulatory machinery may provide a novel weapon to treat hepatocellular carcinoma \[\[[@B27]\] and references there in\].

In summary, ALA has a markedly cytotoxic effect on hepatocarcinoma cell lines at physiological levels, which could be attributed to an enhancement of apoptosis, independent of p53 but due to inhibition of cell cycle regulators CDKs.

Competing interests
===================

None declared

Pre-publication history
=======================

The pre-publication history for this paper can be accessed here:

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