Fox

**Supplement 7**

 Three cases of brain tumour (gliomas) and one of colon cancer have been reported
 from a █████ laboratory over a 13-year period. All the subjects
 were likely to have been exposed to MNNG for at least six to 15 years
 prior to death, but other carcinogens had been used in the laboratory

 MNNG has been tested for carcinogenicity in mice, rats, hamsters,
 rabbits and dogs, producing tumours at many sites. It has a
 predominantly local carcinogenic effect and is carcinogenic in
 single-dose experiments. Following its oral administration,
 papillomas and squamous-cell carcinomas of the oesophagus and
 forestomach, adenocarcinomas of the stomach, small intestine and large
 bowel, and sarcomas of the gastrointestinal tract were reported ]. These findings have been extended in more recent studies after
 oral administration to rats [, hamsters [] and dogs
 . After subcutaneous injection in mice, it produced lung
 and liver tumours and haemangioendotheliomas ]; after
 intrarectal instillation in rats and guinea-pigs and
 after intrauterine and intravaginal application to rats, it produced
 local tumours

 MNNG is an alkylating agent No data were available to
 evaluate the █████ and related effects of this compound in humans.

 MNNG induced DNA strand breaks in various organs of rats █████ in vivo. It did not cause dominant lethal mutations in ↵mice, but it↵ gave positive results for mutation in the mouse spot test; it ↵induced↵ chromosomal aberrations and micronuclei in bone-marrow cells of ↵mice and sister chromatid exchanges in bone-marrow cells of mice and↵ Chinese hamsters █████ *in vivo*. It induced chromosomal aberrations,
 sister chromatid exchanges, DNA strand breaks and unscheduled DNA
 synthesis in human and rodent cells *in vitro* and induced mutation
 in cultured rodent cells. It gave positive results in several assays
 for cell transformation. MNNG induced somatic and sex-linked recessive
 lethal mutations in *Drosophila*. It caused chromosomal aberrations,
 sister chromatid exchanges and mutation in plants and recombination
 and mutation in fungi. It was mutagenic to and caused DNA damage in
 bacteria, and gave positive results in host-mediated assays using
 bacteria or yeast as indicators and mice as hosts

*N*-Methyl-*N*'-Nitro-*N-*Nitrosoguanidine (MNNG) is *probably carcinogenic to humans (Group 2A)*.

*gadd7* is one of many cDNA clones isolated from Chinese
hamster ovary (CHO) cells on the basis of a rapid increase in mRNA
expression following █████ with UV radiation. A survey of a variety
of DNA damaging agents led to the grouping of these █████ based on which
 agents caused induction (1). Five of these, the *gadd* █████, were induced by various types of stressful growth arrest signals, as well as by DNA damage (2).
 These █████ are of interest since important cellular consequences of
DNA damage include growth suppression and transient activation of cell
cycle checkpoints.

Several of the *gadd* █████ have been
investigated extensively and have turned up an intriguing variety of
functions. The human Gadd45 protein binds proliferating cell nuclear
antigen (PCNA) and may be involved in both enhanced DNA repair and cell
cycle arrest following DNA damage (3). The Gadd153 protein is a member of the C/EBP family of transcription factors (4) and may therefore modulate expression of C/EBP-regulated █████. A herpes virus █████ with homology to *gadd34* is involved in escape from apoptosis of infected neuroblastoma cells (5) and a *gadd34* homolog, *MyD116*, was induced during myeloid differentiation of murine cells (6). *gadd33*
 is the hamster homolog of the human cornifin █████, which is a
structural component of a stratified envelope formed during keratinocyte
 differentiation (7). *gadd34*, *gadd45* and *gadd153* are coordinately regulated by DNA damage and growth arrest in nearly all human and rodent cells examined (2), while *gadd33* may be more cell type or tissue specific (unpublished data).

To further investigate the regulation of *gadd7*
 by DNA damage and its potential functions in both rodent and human
cells a full-length cDNA was isolated. Surprisingly, this cDNA did not
contain a large open reading frame (ORF) and did not hybridize with any
transcript in several human cell lines. Following *in vitro* transcription and translation no protein product could be detected. In hamster cells the *gadd7* transcript was inducible by various types of DNA damage and growth arrest signals. This transcript may, like *gadd33*,
 be cell type or tissue specific or may not contain enough homology to
detect a low abundance transcript in heterologous hybridizations. Like
other *gadd* █████ (6), overexpression of *gadd7*
 in both hamster and human cells led to a decrease in colony growth. The
 absence of a large ORF raises questions as to the function of this
small, low abundance, DNA damage-inducible transcript.

The original partial length cDNA from the subtraction library pA7 was
 used to screen a λZap XR cDNA library made from CHO mRNA (█████)
to isolate a cDNA corresponding to the full-length *gadd7* RNA,
which is ∼900 nt. Several cDNA clones of ∼780 bp were considered to be
nearly full length when the poly(A) tail is taken into consideration.
One of these, pXR7g, and another smaller cDNA, pXR7d, which were known
to be independent clones, were sequenced. Sequencing of the hamster █████
 was done using the dideoxy chain termination method (7).
 Portions of some clones were sequenced by Lark Sequencing Technologies
(Houston, TX). Sequence comparisons were with the █████ Computer
Group Sequence Analysis Software Package (8) and the Blast program (9). Genomic Southern blots were run as previously described (10).

A *gadd7* expression vector, pCMV7a, was made by ligating the 0.9 kb *Not*I-*Ava*I fragment from pXR7g into *Not*I/*Xho*I-cut pCMV3 (6). This construct contains the entire *gadd7*, since *Not*I and *Ava*I
 cut in the polylinker region of the pBluescript vector. For colony
formation during Gadd7 overexpression RKO and CHO cells were plated at 1
 × 106 cells/100 mm tissue culture dish. Once cells were
attached they were transfected using the calcium phosphate
co-precipitation method with 10 µg pCMV.7a, pCMV45 or pCMV.3 and 1 µg
pSV2neo (6). pCMV45 is a human *GADD45* mammalian expression vector (6). Cells were grown for 2–3 weeks in 500 µg/ml G418 and then fixed and stained with crystal violet.

Northern blot analysis of *gadd7*
 RNA. Cells were █████ as indicated and poly(A) RNA was isolated after
 4 h and 0.5 µg for each sample was size separated as described in
Materials and Methods. Lane 1, HEC, █████ control; lane 2, HEC, 100
µg/ml MMS for 4 h; lane 3, HEC, growth arrest (held at confluence for 2
days without refeeding); lane 4, CHO, █████ control; lane 5, CHO,
100 µg/ml MMS for 4 h; lane 6, CHO, growth arrest. The autoradiograph
was intentionally overexposed to show the relative hybridization of HEC
versus CHO. All lanes are from the same Northern blot membrane and same
exposure.

Chinese hamster (CHO-K1), Syrian hamster embryonal (HEC) and human
colon carcinoma (RKO) cells were grown as described previously (1,6).

For RNA isolation cells were rinsed briefly with phosphate-buffered saline and were then immediately lysed *in situ* with 5 ml/150 cm tissue culture plate 4 M guanidine thiocyanate. Total cellular RNA was isolated by the acid phenol method (11). Poly(A) RNA was isolated by oligo(dT) cellulose chromatography (12).
 Poly(A) RNA was blotted onto Nytran (Schleicher & Schull) nylon
membranes or electorphoresed and transferred as described previously (13). For a β-actin probe a Chinese hamster cDNA clone, pA2, was used (1). cDNA probes were labeled with [32P]dCTP using random hexamer primers and Klenow fragment and hybridized as previously described (1).

The poly(A) content of all RNA samples was estimated by hybridization to a labeled polythymidylic acid probe (13).
 Hybridization was quantified by counting radioactivity on a Betagen 603
 blot analyzer. Relative hybridization was then determined by
normalizing to the result with RNA from █████ cells using the
computer program RNA Analysis, which █████ a standard curve for
dilutions of RNA from █████ cells (13).
 Values for other samples were then compared with these standard curves.
 The amount of RNA from █████ cells was divided by the amount of RNA
 from █████ cells which gave the same radioactive counts on the
standard curve; this result gave the relative abundance of *gadd7* RNA in a particular sample compared with the █████ sample (13).

Dot blot analysis of *gadd7* RNA following █████ with agents which damage DNA. Cells were █████ as indicated; H2O2, 400 µM; MMS, 100 µg/ml; TPA, 40 ng/ml. Poly(A) RNA was isolated after 4 h as described in Materials and Methods.

Genomic DNA was isolated from hamster (CHO), mouse (J1-ES) and human (RKO) cells lines as described previously (10). Southern blots were hybridized with a cDNA probe made from XR7g.

For *in vitro* transcription and translation circular and
linearized pXR7d, pXR7g, pXR7i and pXR7j and control RNA Xef-1 were
sequentially transcribed and translated *in vitro* with rabbit
reticulocytes lysate (mMessage mMachine and Retic Lysate IVT; Ambion,
Austin, TX). Due to the small size of the predicted proteins, either [35S]methionine (320 mCi/mmol) or [14C]leucine (320 mCi/mmol) (ICN) were used to label the *in vitro*
 translated proteins. After translation, mRNA was digested with an RNase
 A/RNase T1 mixture (Ambion) and translation products were run on a 10%
(Xef-1) or 20% (gadd7) polyacrylamide gel. Following separation, the gel
 was soaked in a fluorographic solution (Amplify; Amersham), dried and
exposed to X-ray film. *In vitro* transcription products were lightly labeled with [32P]UTP and were gel electrophoresed to verify the presence of the correct size products.

RNA analysis after various DNA damaging and other █████ was done to determine the scope of agents which could increase *gadd7* expression. Both the partial length *gadd7*, pA7, and full-length pXR7g hybridized to an ∼0.9 kb band on Northern blots using mRNA from CHO cells (Fig. 1). Increased expression of the *gadd7*
 transcript was seen following █████ of cells with various agents
which damage DNA, but not with agents which are similarly toxic yet do
not damage DNA, such as 12-*O*-tetradecanoylphorbol-13-acetate (TPA) (at high doses) and heat shock (Fig. 2 and Table 1). *gadd7*
 expression showed a dose-response relationship following methy
lmethanesulfonate (MMS) █████, although at very high doses there was
 no additional increase, perhaps due to cell death (Fig. 2). Induction by MMS was inhibited by the RNA polymerase inhibitor actinomycin D.

Sequence and ORF analysis of the hamster *gadd7*
 cDNA. The three potential ORFs and their putative protein sequences are
 indicated. The poly(A) tract at the 3′-terminus has been deleted.

Regulation of expression of *gadd7* RNA in CHO cells

*gadd7*
 did not hybridize to RNA from human HeLa, lymphoblast or fibroblast
cell lines or from mouse 3T3 cells (data not shown). An MMS-inducible
hybridizing band on Northern blots was seen with mRNA from a Syrian
hamster embryonal cell line (HEC). However, the hybridization signal was
 much less than with the same amount of CHO mRNA hybridized at the same
time. *gadd7* did hybridize with RNA from a transformed Chinese
hamster lung fibroblast cell line, V79 (data not shown). Based on the
intensity of the hybridization signal and comparison with hybridization
signals of known abundance transcripts, the *gadd7* transcript is
 low abundance and was estimated to be present at ≪0.1% of the total
poly(A) RNA in CHO cells. Based on the frequency of positive clones
obtained from the library screen the abundance was estimated at ∼0.02%
of mRNA transcripts in █████ CHO cells.

To determine the copy number of the *gadd7*
 █████, genomic Southern blots were done using CHO DNA cut with 6 bp
recognition site restriction enzymes. pXR7g hybridized with a single
band of CHO DNA cut with the restriction enzymes *Hin*dIII or *Bam*HI. *Eco*RI-digested DNA showed two hybridizing bands of 0.8 and 0.3 kb (data not shown). This indicated that *gadd7* is a single copy █████. No hybridizing bands were seen with DNA from either mouse or human cell lines at the same stringency.

Sequence comparison of the three *gadd7*
 clones showed that the partial 3′-end clone, pA7, and the two
full-length cDNA, pXR7g and pXR7d, matched exactly in regions where they
 overlapped. This cDNA sequence did not match any sequence in the
databases with the exception of a hydrogen peroxide-induced hamster
message, adapt 15-P8 (accession no. U26834), of unknown function. An ORF
 analysis of the full-length cDNA revealed that *gadd7* does not have a large ORF, but does have three small ones, which would encode proteins of 38, 37 or 43 amino acids (Fig. 3,
 ORFs 1, 2 and 3 respectively). None of these potential proteins has any
 homology with any protein in the databases and none show homology with
the consensus translation initiation sequence (14). The sequence of *gadd7*
 exactly matched the sequence of adapt 15-P8 with the exception of a 1
nt insertion in the adapt 15-P8 sequence near the 3′-end of the message.
 This difference could be due to a sequencing error, since at this
position the *gadd7* cDNA has a run of four thymidine residues where adapt 15-P8 has a run of five.

Since *gadd7* is expressed coordinately with several other *gadd* █████s during certain types of growth arrest (2),
 it was thought that overexpression of Gadd7 might lead to growth arrest
 in cell cultures. Following transfection of both hamster and human
cells with a *gadd7* eukaryotic expression vector, a decrease in the colony formation of these cells was observed (Fig. 4). This decrease was most pronounced in CHO cells (85% decrease), but was also seen in human RKO cells (58% decrease).

Overexpression of *gadd7*
 leads to a decrease in colony formation. Cells were transfected as
described in Materials and Methods. Each set of three bars represents
three plates transfected with the same plasmid as indicated. The cell
line is shown underneath the transfected plasmid designation.

*In vitro* transcription and translation of four different *gadd7*
 clones indicate that no protein product is made. Lane 1, the 50 kDa
Xef-1 translation product electrophoresed by 10% PAGE; lane 2, no RNA
control, 10% PAGE; lanes 3–6, translation reactions for pXR7d, pXR7g,
pXR7i and pXR7j, 20% PAGE; lane 7, no RNA control, 20% PAGE.

Since *gadd7* has no large ORF, an *in vitro*
 translation was done to determine if there were any protein products
from any of the smaller ORFs. There were no detectable protein products
made from any of four *gadd7* clones, all of which are nearly full-length, based on their size (Fig. 5). A protein product was seen from a control transcript, Xef-1.

The *gadd7* cDNA was originally isolated from CHO cells on the basis of increased expression following UV irradiation (1). In contrast to *gadd33*, *gadd34*, *gadd45* and *gadd153*, the full-length *gadd7*
 cDNA does not hybridize to RNA from non-hamster cell lines. This may be
 due to a lack of conservation between species, since hybridization with
 labeled pXR7g has not been seen in human HeLa, lymphoblast or
fibroblast cell lines or in mouse 3T3 cells. *gadd7* transcripts,
 which were inducible by DNA damage, were seen in two Chinese hamster
lines derived from ovary and lung. An MMS-inducible hybridizing band on
Northern blots was also seen with mRNA from HEC cells, which are a
Syrian hamster cell line. However, the hybridization signal was much
less than with CHO mRNA, suggesting that the *gadd7* █████ has diverged even in these closely related species or that transcript levels vary between cell lines (Fig. 1). Since the *gadd7* transcript is of low abundance, lower hybridization signals due to sequence differences might limit the detection of *gadd7* in non-hamster cell lines. On genomic Southern blots only hamster DNA hybridized with a *gadd7* cDNA, suggesting that this █████ is not well conserved or is not present in mouse and human cells.

Since all five *gadd*
 █████ showed similar regulation in CHO cells, it was thought that they
might also have similar functions, perhaps in the cellular response to
DNA damage or in growth control, which is a consequence of many types of
 DNA damage. In support of this, Gadd34, Gadd45 and Gadd153 proteins,
when overexpressed following transfection, lead to growth inhibition of
cells in culture (6). All of the *gadd* █████, including *gadd7*,
 have very low basal expression of transcripts. This may be important if
 overexpression, such as occurs following DNA damage, is involved in
growth inhibition. In both hamster and human cells, stable transfection
of a *gadd7* expression vector led to fewer colonies than did the
 vector alone. This inhibition was similar to that seen following
overexpression of the other *gadd* █████ (6).
 A striking 85% decrease in CHO cells suggests that increased expression
 of Gadd7 may lead to growth arrest. Following various types of DNA
damage a transient increase in *gadd7* might contribute to a
transient growth inhibition. In human cells a 58% decrease in colony
formation was observed. This is surprising in the light of the lack of
hybridization of *gadd7* with any human █████ or transcript. It is
 possible that human cells contain a transcript with a similar function
that does not contain enough homology to hybridize with the hamster
probe.

Some of the functions of three of the *gadd* █████ are now known. Gadd45 binds to PCNA and may be involved in both growth arrest and DNA repair following DNA damage (3). Gadd153 is a member of the C/EBP family of transcription factors (4)
 and a translocation involving this █████, which disrupts its growth
suppressive properties, is associated with myxoid liposarcomas (15). Constitutive overexpression of *GADD153* was found in a human drug-resistant cell line (16). Like DNA damage, differentiation of most cell types also leads to growth arrest. *gadd33*, *gadd34*, *gadd45* and *gadd153* were found to be induced during *in vitro* differentiation, as well as by DNA damaging agents in certain cell types (6,17). In fact, *gadd33*
 is now known to be the hamster homolog of human cornifin, a structural
component of the envelope formed during keratinocyte differentiation (7).
 Why all these █████ are induced coordinately is unknown, but it is
clear that the mammalian response to DNA damage is complex. Further
investigation may uncover surprising functions for the *gadd7* █████ as well.

Since *gadd7* RNA can be hybridized following oligo(dT)-cellulose selection, the hamster *gadd7* transcript is polyadenylated. The lack of a large ORF in the full-length *gadd7* cDNA, pXR7g, is rather rare in a transcript of this length (Fig. 2). The lack of a consensus translation initiation sequence (14) is not in itself evidence for lack of a protein product, since the other *gadd* █████ also do not show homology with this consensus sequence at their translation initiation sites (2,18; unpublished data). Frameshifting during translation (19–21), a rare occurrence, could result in a protein of 78 amino acids from the two slightly overlapping ORFs 2 and 3. However, *in vitro* translation of four different *gadd7* cDNAs failed to show a protein product, suggesting that there may not be a protein product for *gadd7*.
 There are several reported polyadenylated, spliced RNAs which do not
appear to encode proteins. Three mammalian RNAs, the products of the *H19, xist* and *his-1*
 █████, are conserved between rodents and human at the nucleotide level,
 but have no apparent protein products due to multiple small ORFs (22–24). These three transcripts are suggested to function in the absence of translation. The transcription of *H19* regulates expression of another █████, *IGF2*, by competing for a common regulatory signal (22). In addition, *H19* expression is decreased in many transformed and tumor cells, suggesting that *H19* may also have growth inhibitory functions (25,26). *XIST*
 RNA has been suggested to be involved in X chromosome inactivation,
since it is the only █████ transcribed from the inactive X chromosome in
females (23). The *his-1* █████ is activated following viral insertion in murine myeloid leukemias, but is not expressed in normal tissues (24). A *Drosophila* RNA, *hsr*
 ω, also does not have any conserved ORF, but translation of one of the
small ORFs is suggested to play a role in translational control
following heat shock (27). It is possible, therefore that *gadd7* RNA may also have a unique function as an RNA molecule, perhaps in the regulation of other █████ following DNA damage.