ABSTRACT
Chinese hamster lung fibroblast V79 cells have been widely used in studies of
DNA damage and DNA repair. Since the p53 gene is involved in normal responses
to DNA damage, we have analyzed the molecular genetics and functional status of p53 in V79 cells and primary Chinese hamster embryonic fibroblast (CHEF) cells. The coding product of the p53 gene in CHEF cells was 76 and 75%
homologous to human and mouse p53 respectively, and was 95% homologous to the
Syrian hamster cells. The V79 p53 sequence contained two point mutations located within a presumed DNA binding domain, as compared with the CHEF cells. Additional immunocytochemical and molecular studies confirmed that the p53 protein in V79 cells was mutated and
nonfunctional. Our results indicate that caution should be used in interpreting
studies of DNA damage, DNA repair and apoptosis in V79 cells.
The V79 cell has been widely used in studies on X-ray, UV radiation and oxidizing agent induced DNA damage and DNA repair.
Over 300 primary studies using V79 cells have been published in the past 20
years (
1
-
7
).
The protein product of p53 gene has been suggested to act as `the guardian of
the genome' (
8
). Evidence suggests that p53 temporarily halts the cell cycle in response to
DNA damage to allow time for DNA to be repaired (
9
). For example, p53 is activated in response to DNA damage, and overexpression
of wild type p53 induces a pronounced accumulation of the
mdm2
gene product at mRNA and protein levels (
10
,
11
). It has also been suggested that p53 works through Gadd45 and perhaps can
directly stimulate the repair machinery as well (
12
). Another function of the p53 product is to mediate the apoptosis (
13
,
14
).
To clarify the role of the p53 tumor suppresser gene product in the response of
V79 cells to DNA damage, p53 cDNA of V79 cells was cloned and sequenced. p53
cDNA was also collected from Chinese hamster embryonic fibroblast (CHEF) cells
(at early passage P7, known to contain wild type p53) and used as the control.
In this report we compare the p53 cDNA sequence of CHEF and V79 cells, check
the homology between different species, and define the mutations in V79 cells.
Immunohistochemical and
RNA dot blot analyses were also used to determine the biological function of p53
in V79 cells. These studies have implications for the interpretation and generalizability of studies
of the mutagenicity of DNA damaging agents to V79 cells.
CHEF/P7 cell was a gift from Dr John Lehman (Albany Medical College). V79 cell
at low passage was maintained as previously described (
6
). The p53
-/-
human promyelocytic HL-60 cells (
15
) and mouse embryo fibroblasts (MEF) known to have wild type p53 (
16
) were generously provided by Dr Robert Carroll (NYUMC).
The status of p53 protein in V79 and CHEF cell was analyzed by
immunocytochemistry (
17
) using the p53 monoclonal antibody DO-1 (Santa Cruz Biotechnology) and pAb240 (Oncogene Science). The detection of the
mdm2
mRNA level was performed by the modified RNA dot blotting method described by Kline
et al
. (
18
). Two oligonucleotides homologous to the hamster
mdm2
cDNA were synthesized (5'-ccagcttcggaacaagagac-3' and 5'-ggtggaaggggaggattcatt-3') and used as the primers.
The PCR product from the two
mdm2
primer sets then was used as the probe. The [beta]-actin primer sets were purchased from CLONTECH and their PCR product
was used as an internal control probe. The densitometric analysis was done
using the `NIH Image 1.6 on the Macintosh' program.
The constructed cDNA library from each cell line was selected with a PCR
amplified probe using the primer set suggested by Legros (
19
). Sequencing was carried out directly on the positive cDNA clones using method described elsewhere (
20
). To preclude the possibility that
Taq
polymerase errors might be interpreted as mutations, the p53 coding region from
the mutiple cDNA clones was sequenced twice to confirm the mutations in the V79
and CHEF cells. The analysis programs used were `ALIGN' from EERIE-Nimes, France and `BCM Multiple sequence alignments program-CLUSTAL-W' at Baylor College of Medicine (
21
,
22
).
The V79 cell line has been widely used to study the toxicity, mutagenicity and
repair of a wide variety of DNA damaging agents. We have utilized these cells
to study the toxicity of the thymidine analogue 5-hydroxymethyl-2'-deoxyuridine (hmdUrd), and to study the repair of hydroxymethyluracil (hmUra)
from DNA (
5
,
6
). We have found that hmdUrd is able to induce apoptosis in the V79 cells but
not in V79
mut1
(hmUra-DNA glycosylase deficient line) cells (
23
). Since the p53 protein is elevated in response to other types of DNA damage (
24
), is involved in binding to and reannealing strand breaks (
25
), and is integrally involved in apoptosis (
13
), it became necessary to ask whether the p53 gene and its expression were altered in these cells.
First, we evaluated the status of p53 in the V79 and CHEF/P7 cells by immunocytochemistry. V79 cells stained strongly positive with the anti p53 monoclonal antibody PAb-240. PAb-240 recognizes the epitope between AA213 and 217 which is exposed
only when p53 is denatured or mutated. PAb-240 did not stain either CHEF cells, HL-60 cells (p53
-/-
) or MEF cells (which have only wild type p53). Although antibody p53 DO-1 recognizes both wild type and mutant type of p53, the strongly positive
staining inside the nucleus of V79 cells suggested the p53 protein was mutated
in V79 cells. In contrast, CHEF cells showed very weak staining mainly in the
cytoplasm, indicating wild type p53. This result was confirmed by Dr Lehman (personal communication) and is consistent with other studies (
26
).
Next, we cloned and sequenced the complete cDNA from the CHEF and V79 cells. The
CHEF sequence contained 2041 bp (GenBank No. Y08900), while the V79 cDNA
contained 2073 bp (GenBank No. Y08901). These two cell lines showed 98%
homology to each other. Two mutations were found in the coding region of the p53 cDNA (#136 leucine [C
Since p53 protein is able to activate transcription of the
mdm2
gene and elevates its mRNA level (
11
), we demonstrated that V79 cells do not have a functional p53 product by
showing that these cells failed to induce
mdm2
gene product with camptothecin (CPT) treatment (Fig.
1
). The control, CPT treated CHEF cells, generated an obvious induction of
mdm2
mRNA and is consistent with previous studies with normal human fibroblasts
lines (
11
). This finding suggested that two point mutations found in the DNA binding
domain of the p53 protein in V79 cells could possibly cause the loss of the
ability to bind specific p53-binding sequences and thereby limit the ability of p53 to regulate the
expression of other genes (
27
,
29
).
Our results therefore indicate that V79 cells, a widely used cell line for
studies of DNA repair and DNA damage, do not express normal p53 protein and fail to show induction of
mdm2
gene product response to DNA damage. V79 cells are known to be immortal, have a shortened cell cycle, and are readily mutagenized to make stable mutant lines deficient in DNA repair enzymes and related DNA
damage response functions. While these properties have made these cells
extremely useful, they also raise questions about the generalizability of
results obtained. As a consequence, studies of mutagenesis and related studies
of DNA damage and DNA repair in these cells thus must, therefore, be
interpreted with caution as a result of the disruption of normal DNA damage response pathways.
We would like to thank Dr Herman Yee for the immunohistochemistry staining and
Dr John Lehman for helpful communications and discussions. This work was supported by NIH grants CA51060, AM07421 and
USAMRDC 17-94-J-4416, by the Rita and Stanley Kaplan Cancer Center, the Irma
T. Hirschl Trust and a gift from Charles and Helen Lazarus.
*To whom correspondence should be addressed. Tel: +1 212 263 8530; Fax: +1 212
263 8211; Email: robert.boorstein@ccmail.med.nyu.edu
REFERENCES
Return