Cultures of permanent lines of human promyelocytic cells and their uses for the screening of molecules utilizable in particular in the treatment of leukemias

The invention concerns permanent cell lines of human promyelocyte cells characteristic of acute promyelocytic leukemias, these cells being cytogenetically characterized by a translocation t(15;17), capable of indefinite proliferation in a culture medium. It also concerns the use of these cell lines, particularly for screening molecules capable of inducing cell maturation.

This application was filed under 35 U.S.C. 371 as the national stage of 
PCT/FR92/00173 filed Feb. 25, 1992, and published as WO92/14815 Sep. 3, 
1992. 
Acute promyelocytic leukemia (or APL or also leukemia type 3) is a 
well-defined variety of leukemia, and is cytogenetically characterized by 
a translocation t (15; 17) (q22; q11-12) (Rowley J. D. et al., Lancet 
1:549, 1977). This translocation could play a role in the uncontrolled 
cellular proliferation with blockage of cell maturation which occurs in 
APL. 
In vitro and in vivo studies have made it possible to demonstrate that 
retinoic acid (also designated hereafter as RA) stops the process of 
cellular proliferation of the APL and induces the morphological and 
functional maturation of the promyelocytes towards the granulocyte stage. 
This effect of retinoic acid on the promyelocytes suggests that the 
expression of certain genes intimately implicated in the transduction of 
the RA signal or in the process of maturation of the cells is adversely 
affected by the translocation mentioned above. 
In vitro studies conducted on promyelocytes derived from patients suffering 
from APL have made it possible to demonstrate that the gene coding for the 
alpha receptor of retinoic acid (RAR.alpha. receptor) normally located on 
chromosome 17 has been rearranged by translocation with the PML locus 
normally located on chromosome 15 (de The et al., Nature, 347, pp558-561, 
1990). By using probes corresponding to segments of DNA localized in the 
vicinity of the breaking points of the translocation, it was possible to 
define genomic rearrangements of one or other locus in the patients 
suffering from APL mentioned above. Consequently, the RAR.alpha. and PML 
genes are rearranged in the APLs. These experiments strongly support the 
implication of the alpha receptor of retinoic acid in acute promyelocytic 
leukemias. 
In the light of the importance of the role played by the RAR.alpha. in the 
pathology of the APLs, it would be particularly interesting to be able to 
have available cell lines constituted of promyelocytes of human origin 
which have this translocation t (15; 17) characteristic of the APLs in 
order in particular to screen molecules derived from retinoic acid, or 
other molecules capable of restoring cell maturation without nonetheless 
exhibiting the toxic effects of retinoic acid. 
However, it has hitherto not been possible to maintain a line of human 
promyelocytic cells in culture and, consequently, it has not been possible 
to perform this type of study. 
Although the HL60 line has been called "promyelocytic" (Gallagher R. et 
al., Blood 54:713, 1979), it was subsequently recognized that, on the one 
hand, it is derived from a myeloblastic leukemia characterized by cells 
with a certain degree of maturation (M2) (Dalton W. T. et al., Blood 71; 
242, 1988) and, on the other, it does not contain the above-mentioned 
translocation t (15; 17). 
One of the objectives of the present invention is precisely that of making 
available a cell culture which permits the screening of molecules capable 
of restoring cellular maturation. 
The subject of the present invention is permanent cell lines, characterized 
in that they are constituted essentially of promyelocytic cells 
characteristic of acute human promyelocytic leukemias, these cells being 
cytogenetically characterized by a translocation t (15; 17), and capable 
of proliferating indefinitely in a culture medium. 
The culture medium in which the above-mentioned cells are capable of 
proliferating is advantageously constituted by a synthetic nutrient medium 
(RPMI 1640) and fetal calf serum. 
The cell lines of the invention and their variants are characterized 
particularly in that they possess DNA fingerprints on gel electrophoresis 
identical, wholly or in part, with the DNA fingerprint shown in FIG. 1. 
As an illustration, the DNA fingerprints of the cells constituting the cell 
lines of the invention are obtained by the procedure defined in Nucleic 
Acids Research (1988), vol. 16, No. 9, p. 4161. 
The cell lines of the invention are advantageously characterized in that 
they are either sensitive to retinoic acid, i.e. capable of 
differentiating from the promyelocytic stage towards the granulocyte stage 
under the influence of retinoic acid, or, on the contrary, are resistant 
to retinoic acid and are thus incapable of differentiating under the 
influence of this latter or also are capable of differentiating as a 
result of treatment by retinoic acid associated with one or more reducing 
agents of such differentiation. 
The invention relates to a cell line sensitive to retinoic acid (i.e. the 
majority of the cells of which are capable of differentiating under the 
influence of this latter), and which was deposited with the Collection 
Nationale de Culture de Micro-organismes de l'INSTITUT PASTEUR (CNCM) on 
25 Feb. 1991 under the number I-1045, as well as with the Deutsche 
Sammlung von Mikro-organismen und Zellkulturen GmbH (DSM) on 14 Feb. 1992 
under the number DSM.ACC.2030. 
This cell line was obtained from a patient suffering from an APL, who had 
been treated by chemotherapy and was in a relapse phase. Cells of the bone 
marrow were taken from this patient and placed in culture on a layer of 
cells of the bone marrow stroma (Lanotte et al., J. Cell. Sci. 50:281, 
1981) until an autonomous cell line was established (designated hereafter 
by the expression NB4). This cell line NB4 is characterized in particular 
by the DNA fingerprint shown in FIG. 1, and is constituted mainly of cells 
sensitive to retinoic acid. It forms the subject of a more thorough study 
in the detailed description of the invention which follows. 
The subject of the invention is also a cell line resistant to retinoic acid 
(i.e. the majoity of the cells of which are incapable of differentiating 
under the influence of the latter), this line (designated hereafter by the 
expression NB4R) being obtained by incubation of the previously mentioned 
NB4 line in the presence of all-trans retinoic acid, followed by the 
isolation of the resistant cells and the amplification of these latter 
still in the presence of retinoic acid. 
The invention also relates to a screening procedure for molecules capable 
of restoring cellular maturation, and more particularly of inducing the 
maturation of the promyelocytes towards the granulocyte stage, in 
particular in the context of acute human promyelocytic leukemias, this 
procedure being characterized in that it comprises the placing of a 
specific quantity of the molecule under study in contact with a cell line 
of the invention, and more particularly with a sensitive cell line, 
followed by the detection of possible differentiation of the cells of the 
said line. 
The subject of the invention is also a screening procedure for molecules 
capable of abolishing the resistance which the cells exhibit, and more 
particularly cells characteristic of the acute human promyelocytic 
leukemias, to the effect on differentiation of retinoic acid or other 
compounds normally capable of inducing cell maturation but for which 
phenomena of resistance by the cells concerned may be observed, the said 
procedure being characterized in that it comprises the placing of a 
specific quantity of the molecule under study in contact with a resistant 
cell line such as defined above, followed by the detection of possible 
differentiation of the cells of the said line in the presence of retinoic 
acid or of the other compounds mentioned above. 
The above-mentioned procedure advantageously makes it possible to select 
molecules which are capable of abolishing this phenomenon of resistance 
mentioned above, on the one hand, and of restoring cellular maturation 
under the above-mentioned conditions, on the other. 
In the context of the procedures mentioned above, the detection of the 
differentiation of the promyelocytes of the cell lines of the invention 
towards the granulocytic stage is advantageously carried out by 
application of one or more of the following methods. 
Since this differentiation is expressed in particular by the activation of 
the expression of certain specific genes, the expression products of these 
genes are detected with the aid of specific antibodies, these antibodies 
being labelled if necessary, in particular radioactively or enzymatically. 
When these expression products exhibit an enzymatic activity or when this 
maturation is expressed by a disappearance of an enzymatic activity such 
as myeloperoxidase activity, the detection of the differentiation is 
advantageously achieved with the aid of substrates, labelled if necessary, 
in particular radioactively or enzymatically, on which the enzymatically 
active products expressed, or which are no longer expressed, are capable 
of acting. 
The detection of this cellular differentiation may also be carried out by a 
morphological study of the cells, in particular after staining of the 
latter. As an example of a staining method for the cells, mention may be 
made of the May-Grunwald-Giemsa stain. 
Cellular differentiation may also be assessed according to a functional 
criterion, i.e. a study is made as to whether the differentiated cells are 
functional in the same way as normal granulocytes. As an example of a test 
capable of making possible the detection of functional criteria of normal 
granulocytes, mention should be made of the NBT (Nitro Blue Tetrazolium) 
test (Pick E., Methods Enzymol. 133:407, 1986). 
The response time of the cell lines of the invention to the molecules under 
study when used in the procedures described above varies from several 
hours to several days depending on the method of detection of cellular 
differentiation selected. 
As an illustration, the incubation time necessary before detection of the 
activation of the expression of genes is of the order of 30 minutes to 24 
hours, that necessary prior to detection of the modification of the 
morphological aspect of the cells varies from 15 hours to 5 days, and that 
necessary prior to detection of the functional aspect of the cells varies 
from 3 days to 7 days. 
Advantageously, these screening methods make it possible to select 
molecules capable of being used in the area of the prevention or treatment 
of diseases caused by an abnormal proliferation of the cells, in 
particular malignant tumors in general, and more particularly leukemias, 
especially acute promyelocytic leukemias. 
More particularly in the case when cell lines resistant to retinoic acid 
are used, these methods make possible the selection of molecules capable 
of being used in the context of the prevention of the appearance of 
resistance phenomena directed against the differentiation effect of 
retinoic acid or other compounds normally capable of inducing cell 
maturation of the cells of a patient when this latter is being treated 
with retinoic acid or compounds mentioned above. 
Still in the context of the use of cell lines resistant to retinoic acid, 
these procedures according to the invention make possible the selection of 
molecules capable of being used in the context of the treatment of 
patients in relapse after treatment with retinoic acid or other compounds 
normally capable of inducing cell maturation. 
The invention also relates to the cultures themselves constituted by cells 
of the cell lines of the invention, particularly the NB4 or NB4R cells 
mentioned above, and a nutrient support enabling the cells to proliferate, 
in particular the RPMI 1640 medium supplemented with fetal calf serum 
indicated above. 
Consequently, the subject of the invention is kits for the implementation 
of the screening procedures such as those described above, these kits 
containing: 
a culture such as that described above comprising a cell line according to 
the invention, 
suitable reagents for the detection of the possible differentiation of the 
cells, in particular: 
* antibodies capable of recognizing specifically the polypeptides produced 
by the activation of the expression of certain genes, these antibodies 
being labelled if necessary, in particular radioactively or enzymatically, 
* substrates, labelled if necessary, in particular radioactively or 
enzymatically, on which the enzymatically active products, expressed or 
which are no longer expressed, are capable of acting, 
* enzymes, labelled if necessary, in particular radioactively or 
enzymatically, and capable of acting on substrates produced during 
cellular differentiation, 
* cell stains. 
The cell lines of the invention are also characterized in that the surface 
membranes of the cells constituting them are recognized in particular by 
all of the antibodes directed specifically against antigens of human 
leukocyte membranes of class I (HLA class I), the markers CD13 and CD33 of 
the myeloid cells, the markers CD15 and CD11b of the granulocytes, the 
markers CD9 and CD11B of the monocytes, the marker CD11b of 
alpha-integrin, the marker CD38 of the activated T cells, the marker CD2 
of the T cells, the marker CD4 of the HIV receptor of the T helper cells, 
respectively, or are recognized by some of them only, but are not 
recognized by the antibodies directed against the antigens of human 
leukocyte membranes of class II (HLA class II), the marker CD10 of the 
antigens of the leukocyte surface membrane of acute lymphocytic leukemias 
(CALLA), the markers CD11c, CD14, CD36 of the monocytes, the marker CD36 
of the platelets, the marker CD7 of the immature T cells, the marker CD3 
of the T cells, the beta chain of the IL-2 cell receptor, the marker CD19 
of the B Pan cells, the CD23 marker of the mature B cells, the marker CD34 
of the precursor cells of the lymphocytes and myelocytes, the marker CD41 
of the platelets of group II/IIIa, the marker CD42 of the platelets of 
group IX, the erythrocytes, glycophorin, or are not recognized by some of 
them only. 
The presence of the marker CD4 at the surface of the cells of the invention 
confers on these latter the property of being a model of choice for the 
study of the process of the infection of the cells by the viruses of the 
HIV type. 
The subject of the present invention is also the use of the above-mentioned 
cell lines as positive controls in the context of in vitro diagnostic 
procedures for acute human promyelocytic leukemias or also the use of the 
resistant cell lines described above as positive controls in the context 
of in vitro diagnostic procedures for the possible appearance of cells 
resistant to retinoic acid or other compounds capable of inducing the cell 
maturation during the treatment of a patient with these compounds. 
The invention also relates to kits for the implementation of the in vitro 
diagnostic procedures for acute human promyelocytic leukemias, these kits 
containing a cell line selected from those described above as positive 
control of the presence of cells characteristic of acute human 
promyelocytic leukemias. 
The invention also relates to kits for the implementation of in vitro 
diagnostic procedures for the possible appearance of cells resistant to 
retinoic acid or other compounds capable of inducing cell maturation 
during the treatment of a patient with these compounds, these kits 
containing a resistant cell line selected from those described above as 
positive control of the presence of cells resistant to the compounds 
mentioned above. 
The subject of the present invention is also polypeptides produced by the 
cell lines of the invention, whether they are secreted or not, and 
implicated in the mechanism of autonomous proliferation of the cells of 
these lines. 
These polypeptides, growth factors for example, are obtained from the cell 
lines of the invention, in particular according to the method comprising 
the following steps: 
incubation of the cells of the lines of the invention in a culture medium, 
withdrawal of a sample of the incubation medium and detection of the 
possible presence of one or more polypeptides such as defined secreted 
into the medium with the aid of a proliferation and differentiation test 
carried out on reference hematopoietic cells, or on the lines NB4 and NB4R 
or their variants, 
biochemical separation (chromatography, electrophoresis, etc. . . ) of the 
polypeptide(s) mentioned above. 
These steps described above lead to the production of the natural 
molecule(s). 
The production of the recombinant molecule(s) may be carried out by 
screening an expression library of the lines NB4 and NB4R or a variant of 
these lines producing the desired biological activity, followed by genetic 
cloning and the production of the above-mentioned recombinant molecule(s). 
The subject of the invention is more particularly the growth factor(s) 
derived from the cell lines NB4 and NB4R, and capable of being obtained 
according to the procedure indicated above.

The invention will be illustrated more particularly with the aid of the 
detailed description which follows of the production of the cell line NB4 
and the cell line NB4R. 
I MATERIALS AND METHODS 
a) Cell culture and establishment of the cell line NB4 
A sample of bone marrow was taken from a patient suffering from APL after 
this patient had received treatment with retinoic add. The leukocytes were 
separated by centrifugation on Ficoll-Hypaque and cultured at a 
concentration of 10.sup.4 cells/ml on a nutrient support constituted of 
cells of human bone marrow stroma (Lanotte et al., J. Cell Sci. 50:281, 
1981), with a RPMI 1640 medium (GIBCO, Grand Island, N.Y.) supplemented 
with 12.5% fetal calf serum (FCS), 7.5% horse serum (Flow Lab, Scotland) 
in a plastic bottle at 37.degree. C. in moist air plus 5% CO.sub.2. The 
cultures were maintained by replacing one half of the growth medium each 
week with fresh medium. After 4 to 5 weeks, the adherent layer containing 
regions of proliferating leukemic cells was detached mechanically and the 
entire cell population was transferred without dissociation to a bottle 
containing a fresh nutrient support consisting of cells of bone marrow 
stroma. In this way a progressive enrichment of proliferating APL cells 
was obtained. Development was observed during fourteen weeks, depending on 
the microenvironment. Autonomous growth was then detected and a rapidly 
developing cell population invaded the culture and became a cell line. The 
APL cells thus obtained are designated NB4. The NB4 growth conditions were 
improved by culturing the cells at a concentration of 2.times.10.sup.5 
cells/ml in a RPMI 1640 medium supplemented with 10% FCS only (the 
doubling time varies from 36 to 40 hours). Aliquot quantities of cells 
were frozen at -80.degree. C. 
b) Cytogenetic analysis 
The chromosomes were studied when the cell line had been established and 
after 40 weeks of culture with 2 passages per week. The R bands procedure 
(RHG) was used and the chromosomes were classed according to the 
international nomenclature (ISCN/1985/: An International System for Human 
Cytogenetic Nomenclature) (Harnden D. G., Klinger H. P./eds/Published in 
collaboration with Cytogenet Cell Genet. Basel, Switzerland, Karger, 
1985). 
c) Induction of differentiation 
The NB4 cells (10.sup.5 cells/ml) in RPMI medium containing 10% FCS and 
variable concentrations of all-trans retinoic acid (Sigma) as inducing 
agent were incubated for 1 to 5 days on culture microplates. The 
maturation was assessed by microscopic examination of the enzymatic 
staining on the cytological plates and also by the adhesion of the cells 
of the stroma to the extracellular matrix, fibronectin. The cells were 
stained with the aid of May-Grunwald-Giemsa. The reactions were carried 
out with myeloperoxidase, alpha-naphthyl butyrate esterase, naphthol-ASD 
chloroacetate esterase, alkaline phosphatase and with NBT. 
d) Cell surface markers 
The staining of the cell suspensions in the case of indirect 
immunofluorescence was carried out according to the methods described in 
(Chen Z. et al.: Immunological typing of ALL: Concurrent analysis by flow 
cytometry and immunocytology. Leuk Res. 10:1411, 1986). The 
cytofluorometric analysis was carried out on an EPICS profile 
(Coultronics, Margency, France). A large number of monoclonal antibodies 
was used (cf. above). 
II RESULTS 
a) Establishment, morphology and cytometry of the cell line. 
The APL cells were cultured on a layer of cells of the stroma of bone 
marrow. At the start of the culture a sub-population of cells having 
achieved a certain "cooperation" with the hematopoietic microenvironment 
was selected. The total population, with interaction losses with the cells 
of the stroma, was constituted of hypergranular promyelocytes in the G0/G1 
phase, whereas some leukemic cells strongly associated with the cells of 
the stroma had a mitotic activity and were much less granular with the 
morphology of the blast cells found in the APLs. At the time when the NB4 
cell line was isolated, only the blast cells proliferated. After 9 months 
of culture (i.e. about 155 cell doublings) no significant change was 
observed. 
b) Cytogenetic study 
The cytogenetic analyses were performed at the initial stage of the culture 
when the NB4 cell line was isolated, and after nine months of culture. At 
the initial stage of culture, the 23 metaphases examined were abnormal 
with caryotypic variations from one cell to another. The number of their 
chromosomes varied between 68 and 90, most of them being located in the 
hypotetraploid region, with random losses. 
All of the metaphases have the translocation t (15; 17) (q22; q11-12) 
associated with other rearrangements varying from one cell to another. 
However, a loss of chromosome 19 and a replacement by 19q+, on the one 
hand, and a der (12; ?) (p 12; ?) was present in all of the metaphases 
examined. 
The translocation t (15; 17) present in the first caryotype conducted on 
the patient suffering from APL mentioned above and at the stage of his 
second relapse was observed in all of the metaphases of the established 
cell line. The caryotypic complexity which was observed in the culture of 
bone marrow in the short-term was found in the cell line with a great 
variation from one cell to another. The caryotype was hypotetraploid with 
loss of at least one copy of the chromosomes 8, 11 and 14. A 
representative caryotype may be summarised in the following manner: 
80-87, XXX, -X, -3, -8, -9, -10, -12, -14, -14, -18, -19, -19, der (12), t 
(12;?) (p12;), t (15; 17) (q22; q11-12), t (15; 17), der (19) t (19; ?), + 
variable markers. 
In view of this variation, it is difficult to establish the minor 
differences between the caryotypes of the "fresh" cells and the caryotypes 
of the cells of the NB4 line. However, it was possible to detect a new 
marker der (12) in the cell line whereas it was not present in the "fresh" 
cells. The rearrangement of the chromosome 19, resembling HSR (19) (q13), 
which is found in certain metaphases of the "fresh" cells, was apparently 
selected during the establishment of the cell line since it was present in 
all of the metaphases, most of the time in duplicate. 
The caryotype of the NB4 line is shown in FIG. 2. 
c) Immunocytological analysis 
The cell surface markers of NB4 were analysed (cf.above) and monitored for 
9 months, but no significant change was detected. It was thus determined 
that NB4 expresses specific markers at the granulocytic stage, but also 
lymphoid cell markers such as CD2, CD4 and a monocyte marker CD9. The 
percentages of the positive cells compared with the myeloid markers (73% 
to 89%) and T cell markers (73% for CD4) suggest that the characteristics 
of the two lines are expressed simultaneously. CD9 is expressed by 73% of 
the cells, whereas the cells are clearly negative with respect to the 
other markers associated with the monocyte such as CD14 and CD36. The 
expression of these antigens might be associated with activations of genes 
linked to the alterations of multiple caryotypes which were found. 
d) Retinoic acid induces maturation 
NB4 cells were treated with trans retinoic acid (1 umol/l) for six days. 
The cessation of cellular proliferation occurred 48 hours later; 
morphological maturations accompanied by modifications of the surface 
antigens and functional markers can be detected. A large increase in the 
marker correlated with alpha-integrin, CD11b (Arnaout M. A.: Structure and 
function of the leukocyte adhesion molecules CD11 CD18. Blood 75:1037, 
1990) was observed; CD11c, which is absent from blast cells is strongly 
expressed on 75% of the differentiated cells; these changes are associated 
with a marked increase in the adhesion of the cells to the extracellular 
matrices. The production of superoxide and hydrogen peroxide, and the 
reduction of the NBT were analysed quantitatively by ELISA and by 
cytochemical methods; a large increase in the positivity of the NBT test 
indicates a potential and microbial capacity confirming the morphological 
maturation induced by the retinoic acid. 
III ANALYSIS OF THE RESULTS 
The NB4 cell line is the only permanent line exhibiting a translocation t 
(15; 17) which has been established from leukemic cells of a patient 
suffering from APL. 
The cells derived from human APLs (M3) exhibit a remarkable manner of 
proliferating in vitro which is the probable reason for the failures 
hitherto to obtain a permanent cell line. It may be concluded from these 
failures: (1) that the APL cells have a very low proliferation potential 
in vitro. The analysis of the cell cycle by means of cytometry shows a 
cessation of the proliferation in the G1 phase (or possibly in phase G0) 
for several weeks, during which the cells become hypergranular and exhibit 
an intense myeloperoxidase reaction. However, the APL cells survive in 
culture, whereas many cellular types of leukemias die; that suggests that 
these cells do not require survival factors, as distinct from normal 
promyelocytes or cell lines dependent on factors. (2) none of the 
hematopoietic growth factors tested so far (including G-CSF) leads to in 
vitro proliferation for more than 3 to 4 cycles. (3) the APL cells undergo 
cell-cell or cell-matrix interactions with the cells of the stroma of the 
bone marrow. Some APL cells associated with the micro-environment (1 to 10 
per 10.sup.5 cells) proliferate. 
In the light of what has just be stated it might be expected that APL cells 
co-cultured with bone marrow stroma for several months could lead to the 
selection of cells capable of proliferating. The authors of the present 
patent application have put forward the hypothesis that the isolation of a 
permanent APL cell line depends on a second event responsible for the 
autonomy of proliferation and that its probability should be considerably 
increased in a population undergoing cyclic growth as compared to a 
population arrested at stage G1. This strategy was adopted in order to 
isolate the NB4 cell line. The primary culture of leukemic cells on a 
layer of stroma cells provides a favourable environment for cell 
proliferation. It cannot be asserted that an additional mutation is 
responsible for the appearance of autonomous leukemic cells which develop 
in the culture of APL cells depending on the microenvironment. It should 
be emphasized that an additional marker der (12) is present in the cell 
line but cannot be detected in the "fresh" cells. On the other hand, a 
self-renewing cell population might already be present in the bone marrow 
of the patient and would be capable of being selected in vitro. Several 
observations support this latter hypothesis: (1) Among the multiple 
chromosomal changes in the blast cells of the patient, which are perhaps 
partially related to prior treatments, some rearrangements favouring in 
vitro proliferation may have been selected. (2) The NB4 cells have 
conserved morphological characteristics of a minority population of blast 
cells which was also found in the patient at the time of his relapse. 
The retinoic acid induces a rapid maturation of the NB4 cells from the 
morphological and functional point of view; after 3 days of continuous 
treatment with retinoic acid cell proliferation is no longer detectable. 
The maturation of the NB4 cells with other inducers such as phorbol 
esters, dimethyl sulfoxide, cyclic nucleotides and corticosteroids never 
equals in intensity and rapidity the maturation induced by retinoic acid. 
FIG. 1 shows the DNA fingerprint of the NB4 cell line, and which is 
obtained by digestion of the genome of the cells with HinfI, followed by 
migration of the DNA fragments obtained in gel electrophoresis and 
detection of fragments with the aid of the probe M13 (cf. Nucleic Acids 
Research mentioned above). 
FIG. 2 shows the caryotype of the NB4 cell line, the arrows indicate the 
rearranged chromosomes. 
IV ESTABLISHMENT OF THE NB4R LINE 
Cells of the initial isolate of the human promyelocytic line NB4 were 
incubated in the continuous presence of all-trans retinoic acid: after 
several months of culture, some resistant cells were isolated and 
amplified constantly in the presence of retinoic acid. This isolate has 
made it possible to isolate a NB4 cell line resistant to retinoic acid 
(NB4R line). The resistant cells constitute an autonomous line, permanent 
in the same way as the sensitive NB4 line and which can be maintained 
under the same conditions. It is interesting to note that this resistance 
corresponds to a commonly observed clinical fact, namely the relapse after 
a quite prolonged remission following treatment with retinoic acid. The 
patient from whom these lines are derived was himself in relapse after 
chemotherapy and treatment by retinoic acid. Hence it is possible to say 
that the two isolates NB4 and NB4R, one sensitive to retinoic acid, the 
other resistant, represent two parts of this disease. These cells make it 
possible to study the mechanism of acquisition of resistance to the 
treatment of promyelocytic leukemias by retinoic acid. This 
complementarity reinforces the interest in the tool which these lines 
constitute for the study of the effects of the therapeutic agents and for 
the research of agents to prevent (or abolish) resistance to these agents. 
The resistant line constitutes the only existing biological model of a 
promyelocytic line with t (15; 17) enabling molecular screening to be 
carried out in order to discover novel medicines active on the cells of 
patients in relapse after treatment with retinoic acid.