Two prostate carcinoma cell lines, DU-145 and PC-3, were examined for abnormalities in the retinoblastoma (Rb) and the p53 putative tumor suppressor genes. We found an abnormal Rb gene product in DU-145 using Western blot analysis. Polymerase chain reaction amplification followed by direct DNA sequencing demonstrated a base substitution mutation that generates a stop codon in exon 21. On Northern, Southern, and Western blot analysis, the p53 gene and its product appear to be normal in DU-145. PC-3, however, failed to demonstrate expression of either the p53 transcript on Northern blot analysis or the p53 protein on Western blot analysis, while the Rb gene products appeared to be normal on both Northern and Western blot analysis. This work extends the correlation between abnormal expression of putative tumor suppressor genes and human malignancies.

The recombinant shuttle vector pSV2gpt was introduced into V79 Chinese hamster cells, and stable transformants expressing the Escherichia coli gpt gene were selected. Two transformants carrying tandem duplications of the plasmid at a single site were identified and fused to simian COS-1 cells. Plasmid DNA recovered from the heterokaryons was used to transform a Gpt-derivative of E. coli HB101, and the relative frequency of plasmids carrying a mutation in the gpt gene was determined. The high frequency of Gptplasmids (ca. 1%) was similar to that observed when plasmid was recovered from COS-1 cells which had been transfected with pSV2gpt. Most of the mutant plasmids had rearrangements in the region containing the gpt gene.Experimental systems utilizing the Escherichia coli lacI gene and the bacteriophage lambda cI gene have generated a great deal of information concerning both the base specificity and site specificity of mutagens in E. coli (16 Intact plasmid DNA also has been rescued from mammalian cells transformed with SV40-based shuttle vectors after fusion of the transformants with COS cells (3,6). In one study (6) it was observed that when transformants carrying tandem duplications of plasmid DNA were fused with COS cells, all of the rescued plasmid molecules had a normal restriction digest pattern. If intact plasmid could be rescued from transformants of this type, it might be possible to treat such transformants with mutagen, transform bacterial cells with plasmid rescued by COS cell fusion, isolate bacterial transformants containing the mutant gene, recover the mutant gene, and characterize the mutation.In an attempt to develop such a system, we have transformed a V79 Chinese hamster cell line with the SV40-based shuttle vector pSV2gpt (17) Transformation of mammalian cells. Culture dishes (35 mm) were inoculated with 1 x i05 to 2 x 1 743R cells. Twenty-four hours later the dishes were treated with 75 ng of pSV2gpt DNA by the calcium-phosphate precipitation method of Graham and van der Eb (11) as modified by Lowy et al. (14). On the next day, the cells from each 35-mm dish were placed in a 100-mm dish containing medium supplemented with 0.1 mM hypoxanthine, 0.4 mM aminopterin, and 16 F.M thymidine (HAT medium [13]). Approximately 2 weeks later a single colony was isolated from each dish and grown into a mass culture in HAT medium.Preparation of cellular DNA and blot analysis. Cells grown in HAT medium were harvested from two 150-mm culture dishes, and high-molecular-weight DNA was prepared by the method of Blin and Stafford (2). DNA (5 ,ug) from each sample was digested to completion with restriction enzymes according to the recommendation of the manufacturer (New England Biolabs). The digested DNAs were electrophoresed through a 0.7% agarose gel along with lambda Hindlll markers. The gel was then stained with ethidium bromide and visualized under UV light before denaturation, neutralization, and transfer to nitrocellulose (21). 32P-labeled pSV2gpt DNA was prepared by nick translation to a specific activi...

A retroviral shuttle vector was constructed by introducing the Escherichia coli xanthine (guanine) phosphoribosyltransferase gene (gpt) into the pZip-NeoSV(X)1 vector [Cepko, C. L., Roberts, B. E. & Mulligan, R. C. (1984) CeU 37, 1053-1062. This vector was packaged into infectious virus which then was used to infect a hypoxanthine (guanine) phosphoribosyltransferase-deficient mouse cell line. Cell lines that expressed the gpt gene were isolated, and it was found that these cells contained a single integrated copy of the vector in a proviral form. Treatment of these cell lines with either ethyl methanesulfonate or BrdUrd produced a >10-fold increase in the frequency of 6-thioguanine-resistant (Sgur) mutants. Intact gpt genes have been recovered from a number of Sgur cell lines after COS cell fusion and introduced into E. coli as part of a plasmid. The complete DNA sequences of three mutant genes have been determined. Two of the mutant genes have a single base substitution, whereas the third has a 34-base-pair deletion. This system should be valuable for analyzing mutagenic specificity and the molecular mechanisms of chemical mutagenesis in mammalian cells. A potentially important feature of the system relative to other shuttle-vector systems is that the mutations are induced in genes integrated into mammalian chromosomes rather than in genes existing as part of autonomously replicating plasmids."Shuttle vectors" capable of replication in both animal cells and bacteria have been used to study mutagenesis in mammalian cells (1-7). The The selectable gene used in this study is the E. coli gpt gene, which codes for xanthine (guanine) phosphoribosyltransferase (XPRTase). This gene was chosen because there are selective systems for and against XPRTase activity in both bacteria and mammalian cells. The gpt gene was inserted into the retroviral shuttle vector pZip-NeoSV(X)1 (8), which can be packaged into a virus that integrates into chromosomal DNA of infected cells. Also, the vector sequences can be efficiently recovered from infected cells and introduced into E. coli as plasmids. In this paper we describe a system that uses this retroviral shuttle vector to rescue mutant gpt genes from mammalian cells, and we have sequenced three mutant genes recovered after mutagenesis. MATERIALS AND METHODSCell Culture, DNA Transfection, and Virus Infection. Line A9 is a hypoxanthine (guanine) phosphoribosyltransferase (HPRTase)-deficient derivative of mouse L cells (9). COS-1 cells (10) were provided by K. Subramanian and psi-2 cells (11) by R. Mulligan. The basic cell culture medium was Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum; psi-2 cells were grown in medium supplemented with 10% calf serum. HAT medium (12) is supplemented with 0.1 mM hypoxanthine, 0.4 mM aminopterin, and 16 AM thymidine. Selections for G418 (13), 6-thioguanine (Sgu), and ouabain resistance were carried out in medium supplemented with 1 mM G418, 36 pzM Sgu, or 1 mM ouabain, respectively.A retroviral shuttle vector ...

The relationship between bromodeoxyuridine (BrdUrd) mutagenesis in mammalian cells and the effects of BrdUrd on deoxyribonucleoside triphosphate pools was analyzed. It was found that the exposure of Syrian hamster melanoma cells to mutagenic concentrations of BrdUrd resulted in the formation of a large bromodeoxyuridine triphosphate (BrdUTP) pool, which remained at a high level for several days. In contrast, the size of the deoxycytidine triphosphate (dCTP) pool dropped rapidly after the addition of BrdUrd, reached a minimum at about 6 h, and then expanded gradually to nearly its original level over the next 3 days. The addition of lower concentrations of BrdUrd, which had less of a mutagenic effect, resulted in the formation of a smaller BrdUTP pool and a slightly smaller drop in the dCTP pool. When a high concentration of deoxycytidine was added at the same time as a normally mutagenic concentration of BrdUrd, the drop in the dCTP pool was prevented, as was BrdUrd mutagenesis. In all of these experiments, mutagenesis was related to the ratio of BrdUTP to dCTP in the cells. In addition, it was shown that mutagenesis occurred primarily during the first 24 h of BrdUrd exposure, when the BrdUTP/dCTP ratio was at its highest level. It appears that there is a critical ratio of BrdUTP to dCTP that must be attained for high levels of mutagenesis to occur and that the extent of mutagenesis is related to the ratio of the BrdUrd and dCTP pools.Recent studies from our laboratory have suggested that mutagenesis by 5-bromodeoxyuridine (BrdUrd) in mammalian cells involves perturbations of deoxyribonucleotide metabolism. These studies have demonstrated that (i) the extent of mutagenesis is determined by the concentration of BrdUrd to which the cells are exposed rather than by the amount of BrdUrd incorporated into nuclear deoxyribonucleic acid (DNA); (ii) deoxycytidine (dCyd) can suppress BrdUrd mutagenesis without decreasing the amount of BrdUrd incorporated into DNA; and (iii) thymidine (dThd), deoxyguanosine, and deoxyadenosine can stimulate BrdUrd mutagenesis without increasing the amount of BrdUrd incorporated into DNA (5, 9, 10). The triphosphates of BrdUrd and of the three deoxyribonucleosides which stimulate BrdUrd mutagenesis are all known to inhibit the ribonucleotide reductase-catalyzed reduction of cytidine diphosphate to deoxycytidine diphosphate (13,15,18). Thus, agents which inhibit synthesis of dCyd nucleotides stimulate BrdUrd mutagenesis, whereas dCyd itself suppresses mutagenesis. These results suggest that the perturbation of dCyd metabolism plays a key role in BrdUrd mutagenesis in mammalian cells.The experiments in this study were designed to assess directly the relationship between BrdUrd mutagenesis and nucleotide metabolism by monitoring the changes in deoxyribonucleoside triphosphate (dNTP) pools during BrdUrd mutagenesis and dCyd suppression of mutagenesis. The deoxycytidine triphosphate (dCTP), bromodeoxyuridine triphosphate (BrdUTP) and thymidine triphosphate (TTP) pools were measured at times aft...

By using a shuttle vector system developed in our laboratory, we have carried out studies on the molecular mechanism by which 5-bromodeoxyuridine (BrdUrd) induces mutations in mammalian cells. The target for mutagenesis in these studies was the Escherichia coli gpt gene that was contained within a retroviral shuttle vector and integrated into chromosomal DNA in mouse A9 cells. Shuttle vector-transformed cells expressing the gpt gene were mutagenized with BrdUrd and cells with mutations in the gpt gene were selected.Shuttle vector sequences were recovered from the mutant cells, and the base sequence of the mutant gpt genes was determined.The great majority of the BrdUrd-induced mutations involving single-base changes were found to be GC -> A-T transitions. We have shown that mutagenesis by BrdUrd depends upon perturbation of deoxycytidine metabolism. Thus, the current results suggest that BrdUrd mutagenesis involves mispairing and misincorporation of BrdUrd opposite guanine in DNA, driven by nucleotide pool perturbation caused by BrdUrd and the resulting imbalanced supply of triphosphates available for DNA synthesis. The results also revealed a very high degree of sequence specificity for the BrdUrd mutagenesis. BrdUrdinduced G-C -> APT transitions occurred almost exclusively in sequences with two adjacent guanine residues. Furthermore, in ,9O% of the cases, the guanine residue involved in mutation was the one in the more 3' position.Our laboratory has shown that mutagenesis by the thymidine (dThd) analog 5-bromodeoxyuridine (BrdUrd) transitions (4-6).We have studied the molecular mechanisms of BrdUrd mutagenesis in mammalian cells, by using a retroviral shuttle vector system developed in our laboratory (7). As the target for mutations, the shuttle vector contains the Escherichia coli gpt gene, coding for the enzyme xanthine guanine phosphoribosyltransferase (5-phospho-a-D-ribose-l-diphosphate: xanthine phosphoribosyltransferase; EC 2.4.2.22). The shuttle vector was introduced into mouse A9 cells and a transformed line (A9I-2) was isolated that contains a single copy of the vector integrated into chromosomal DNA. Since A9 cells are deficient in the enzyme hypoxanthine guanine phosphoribosyltransferase (IMP-pyrophosphate phosphoribosyl-transferase; EC 2.4.2.8) (8), transformants with mutations in the gpt gene can be selected with 6-thioguanine (sGua). For sequencing, the gpt genes from mutant A9I-2 cells can be recovered by fusion with monkey COS cells (9).In the present study, the gpt genes were recovered and sequenced from a large number of BrdUrd-induced mutants of A91-2 cells. We have used this shuttle vector system to analyze mutations that occurred spontaneously or were induced by ethyl methanesulfonate (EtMes) (10, 11). Mutational studies with various shuttle vector systems also have been carried out by other laboratories (12-17). Our system differs from those used in the other studies in that the vector in our system is integrated into chromosomal DNA and clonal selection for cells with mutant ge...