Biomedical subjects

J J McCormick

Publications and source records attributed to J J McCormick.

At least 37 records · Page 2Linked to original sources

PubMed1995

Site-specific rates of excision repair of benzo[a]pyrene diol epoxide adducts in the hypoxanthine phosphoribosyltransferase gene of human fibroblasts: correlation with mutation spectra.

When populations of repair-proficient diploid human fibroblasts were treated with (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) during early S phase, just as the hypoxanthine phosphoribosyltransferase gene (HPRT) was being replicated, 5% of the induced base substitutions were found at nt 212, and 5% of the substitutions were found at nt 229 in exon 3. However, when the population was treated in early G1 phase to allow at least 12 hr for repair before the onset of S phase, 21% of the substitutions were found at nt 212, and 10% were found at nt 229. No such cell-cycle-dependent difference in distribution of base substitutions occurred in excision-repair-deficient cells. To test whether the increase in the relative frequency of mutations resulted from inefficient repair at these sites, we adapted ligation-mediated PCR to measure the rates of removal of BPDE adducts from individual sites in exon 3 of the HPRT gene. Cells were treated with 0.5 microM BPDE in early G1 phase and harvested immediately or after 10, 20, and 30 hr for repair. the nontranscribed strand of exon 3 was analyzed for the original distribution of adducts and those remaining after repair, using Escherichia coli UvrABC excinuclease to excise the adducts and annealing a 5' biotinylated gene-specific primer to the DNA and extending it with Sequenase 2.0 to generate a blunt end at the site of each cut. A linker was ligated to the blunt end, and the desired fragments were isolated from the rest of the genomic DNA by using magnetic beads, amplified by PCR, and analyzed on a sequencing gel. The distribution of fragments of particular lengths indicated the relative number of BPDE adducts initially formed or remaining at specific sites. The rates of repair at individual sites varied widely along exon 3 of the HPRT gene and were very slow at nt 212 and 229, strongly supporting the hypothesis that inefficient DNA repair plays an important role in the formation of mutation hotspots.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed1994

Kinds and locations of mutations induced in the hypoxanthine-guanine phosphoribosyltransferase gene of human T-lymphocytes by 1-nitrosopyrene, including those caused by V(D)J recombinase.

The detection of an increase in the frequency of mutants in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene of circulating T-cells has been proposed as a method to evaluate the biological effects of human exposure to environmental mutagens. We exposed adult human T-cells in vitro to 1-nitrosopyrene (1-NOP), a partially reduced metabolite of 1-nitropyrene, a ubiquitous environmental carcinogen. In populations of T-cells from two unrelated donors, a dose of 1-NOP that reduced survival to 40% of the untreated cells increased the HPRT mutant frequency 6 to 7 times over the background frequency of 5 x 10(-6). The coding region of 35 independent mutants was amplified by polymerase chain reaction and sequenced. Single base substitutions were found in 63% of the mutants (22 of 35). These were distributed randomly throughout the gene. Most of the substitutions (82%) involved G-C base pairs, mainly G.C-->A.T transitions and G.C-->T.A transversions. Fifteen mutants were lacking one or more exons; 9 of the 15 were lacking exons 2 and 3. Examination showed that at least four of the latter had resulted from V(D)J recombinase acting illegitimately to recombine sites located in introns 1 and 3 of the HPRT gene. T-cells from a second unrelated donor were exposed to 1-NOP and 38 additional independent mutants were analyzed. The results indicated that such mutations occurred at a frequency of 2.4 x 10(-6) compared to a background frequency of less than 0.3 x 10(-6). This recombinase, which plays an important role in leukemogenesis, is normally present in developing, but not mature, B- and T-cells such as those used here as target cells for 1-NOP. The present study is the first report showing that exposure to an environmental carcinogen can cause mutations induced by the action of this enzyme.

Amino Acid Sequence↗

PubMed1994

Failure of infinite life span human cells from different immortality complementation groups to yield finite life span hybrids.

The observation that fusion of infinite life span cells with finite life span cells produces hybrid cells with finite life spans led to the conclusion that an infinite life span in culture is a recessive trait resulting from loss of the function of a gene or genes that contribute to an active program for cellular senescence. Furthermore, finding that certain pairs of infinite life span cells, when fused to one another, can complement each other to yield finite life span hybrids allowed 30 infinite life span cell lines to be assigned to four immortality complementation groups (Pereira-Smith and Smith, 1988, Proc. Natl. Acad. Sci. U.S.A., 85:6042). In the present study, we fused a chromosomally stable, near diploid, morphologically normal, infinite life span cell strain, designated MSU-1.1, with its normal, finite life span, precursor cell strain and obtained finite life span hybrids, as expected if infinite life span in culture is a recessive trait. However, 14 of the 14 hybrids from our fusions of MSU-1.1 cells with representative cell lines from each of the four immortality complementation groups, and 38 of the 39 hybrids from our fusions of infinite life span cells that have been reported to complement each other, failed to exhibit finite life spans. This result suggests that infinite life span cells cannot complement each other to yield finite life span hybrids. In examining this unexpected result, we obtained evidence that long-term dual drug selection can be deleterious to hybrid cells even though they carry resistance markers for both drugs, indicating that the cell death of such hybrids observed in other studies may have resulted from the cytotoxic effect of long-term drug selection, rather than from senescence.

Cell Death↗

PubMed1994

v-sis oncogene-induced transformation of human fibroblasts into cells capable of forming benign tumors.

Normal human fibroblasts do not synthesize platelet-derived growth factor (PDGF), but they respond to its mitogenic action. In contrast, cells from several human fibrosarcomas have been shown to synthesize PDGF at significant levels. To investigate the possible role of PDGF expression in the development of tumors of mesenchymal origin in humans, we transfected a plasmid carrying the v-sis oncogene and a selectable marker into an infinite lifespan, non-tumorigenic human fibroblast cell strain, MSU-1.1. The v-sis gene codes for a protein homolog of PDGF-B. Of the six independent drug-resistant transfectants clonally isolated, three expressed a relatively low level of v-sis mRNA and protein, grew to a saturation density only 2-2.5 times higher than MSU-1.1 cells, did not form colonies in agar, were not growth factor independent and did not form tumors. The other three expressed v-sis mRNA and protein at a high level, grew to a very high saturation density (> 750,000 cells/cm2), replicated in medium lacking exogenous growth factors almost as rapidly as with 10% serum, formed large colonies in 0.33% agarose and, when injected into athymic mice, formed tumors that grew rapidly. Tumors that were removed after 6 weeks were classified as benign (fibromas). However, several of the tumors that were left in the animals for 6 months developed focal areas of cells showing features of poorly differentiated spindle cell or round cell sarcomas. Similarly, cells isolated from a very large sized agarose colony formed by one of the other v-sis strains expressed very high levels of v-sis mRNA and protein and formed large, very fast growing benign tumors. Re-injection of cells from the tumors yielded tumors composed of two distinct areas: spindle cell fibromas and high grade sarcomas. These results suggest that if mesenchymal cells in the body were induced to express their PDGF-B gene, this could lead to the formation of benign tumors of mesenchymal origin. The system described can serve as a model for studying the mechanisms of formation of such tumors in humans and their progression to the malignant state.

Animals↗

PubMed1994

Analysis of the multistep process of carcinogenesis using human fibroblasts.

Normal human cells in culture have never been neoplastically transformed by carcinogen exposure. One possible explanation is that the life span of such cells is too short for them to acquire the necessary changes. To test this hypothesis, we needed normal human cells with a greatly extended or an infinite life span. We transfected the v-myc gene and a selectable marker into normal human fibroblasts, identified a drug-resistant clone expressing v-myc protein, and passaged the progeny of the clone until they senesced. A few cells continued to proliferate and gave rise to a diploid, infinite life span cell strain, MSU-1.0, that has normal growth control and is nontumorigenic in athymic mice. Analysis showed that one more genetic change, in addition to unregulated expression of the v-myc gene, was involved in generating MSU-1.0 cells. They spontaneously gave rise to a variant strain, designated MSU-1.1, that grows more rapidly and is less dependent on exogenous growth factors. Analysis showed that at least two additional changes were involved in generating this cell strain. It has a stable karyotype composed of 45 chromosomes, including two markers. Transfection of specific oncogenes was used to determine the number and nature of additional changes required to transform MSU-1.1 cells into malignant cells. Analysis indicated that two changes were involved, but no change in karyotype. Exposure of MSU-1.1 cells to a single carcinogen treatment, followed by selection for cells with the characteristics of oncogene-transformed MSU-1.1 cells also yielded malignant human cells. We conclude that malignant transformation of normal human fibroblasts requires six or seven genetic changes, some of which involve suppressor genes.

Animals↗

PubMed1994

Malignant transformation of a human fibroblast cell strain by transfection of a v-fes oncogene but not by transfection of a gag-human c-fes construct.

To determine whether the human c-fes gene, a homologue of the feline v-fes oncogene, can play a role in the malignant transformation of human fibroblasts, we transfected a near-diploid, infinite life span, growth factor-independent, human fibroblast cell strain, MSU-1.2, with plasmids carrying a fes gene along with a selectable marker. The fes gene was either the v-fes oncogene from the Gardner-Arnstein strain of feline sarcoma virus or a chimeric construct in which 835 base pairs representing exons 10-19 from the human c-fes proto-oncogene had been substituted for the corresponding feline sequence in the v-fes oncogene. The transfected cells were selected in appropriate medium, and a number of drug-resistant clones were isolated, and the progeny cells were assayed for fes expression by immunoprecipitation analysis. Six independent clones that expressed the v-fes protein and four that expressed the gag-c-fes protein were further characterized. The former exhibited anchorage independence and formed progressively growing, invasive, spindle cell sarcomas in athymic mice after only a short latency period. The latter strains were not anchorage independent and did not form tumors in athymic mice. These results show that the v-fes oncogene can malignantly transform an infinite life span human fibroblast cell strain, but the human c-fes gene cannot.

Animals↗

PubMed1993

Fidelity of DNA replication by extracts of normal and malignantly transformed human cells.

To test the hypothesis that a mutator phenotype may be associated with carcinogenesis (L. A. Loeb, Cancer Res., 51: 3074-3079, 1991), we have compared the fidelity of double-stranded DNA replication and the efficiency of mismatch repair in extracts from normal diploid and malignantly transformed human cells. Included was a diploid fibroblast strain and its transformed derivative, as well as a second diploid fibroblast strain and HeLa cells. The fidelity of DNA replication by cytoplasmic extracts in the presence of simian virus 40 large tumor antigen (SV40 T-antigen) was measured using a forward mutagenesis assay. The replicated DNA consisted of double-stranded M13 mp2 DNA containing the SV40 origin of replication and the lacZ alpha complementation gene as a target sequence for scoring mutations. T-antigen-dependent replication was detected in all cell extracts, with those from transformed cells having the greatest activity. No differences in replication fidelity were detected between normal and transformed cell extracts. Using a heteroduplex containing a G.G mispair, we also detected mismatch repair activity in the cell extracts, including efficient repair in extracts from malignantly transformed cells. While these data do not eliminate the possibility that a mutator phenotype may be associated with carcinogenesis, they do suggest that genetic instability associated with transformation does not involve reduced fidelity of replication of undamaged DNA or reduced mismatch repair efficiency.

Cell Line, Transformed↗

PubMed1993

Evidence from mutation spectra that the UV hypermutability of xeroderma pigmentosum variant cells reflects abnormal, error-prone replication on a template containing photoproducts.

Xeroderma pigmentosum (XP) variant patients are genetically predisposed to sunlight-induced skin cancer. Fibroblasts derived from these patients are extremely sensitive to the mutagenic effect of UV radiation and are abnormally slow in replicating DNA containing UV-induced photoproducts. However, unlike cells from the majority of XP patients, XP variant cells have a normal or nearly normal rate of nucleotide excision repair of such damage. To determine whether their UV hypermutability reflected a slower rate of excision of photoproducts specifically during early S phase when the target gene for mutations, i.e., the hypoxanthine (guanine) phosphoribosyltransferase gene (HPRT), is replicated, we synchronized diploid populations of normal and XP variant fibroblasts, irradiated them in early S phase, and compared the rate of loss of cyclobutane pyrimidine dimers and 6-4 pyrimidine-pyrimidones from DNA during S phase. There was no difference. Both removed 94% of the 6-4 pyrimidine-pyrimidones within 8 h and 40% of the dimers within 11 h. There was also no difference between the two cell lines in the rate of repair during G1 phase. To determine whether the hypermutability resulted from abnormal error-prone replication of DNA containing photoproducts, we determined the spectra of mutations induced in the coding region of the HPRT gene of XP variant cells irradiated in early S and G1 phases and compared with those found in normal cells. The majority of the mutations in both types of cells were base substitutions, but the two types of cells differed significantly from each other in the kinds of substitutions, but the two types differed significantly from each other in the kinds of substitutions observed either in mutants from S phase (P < 0.01) or from G1 phase (P = 0.03). In the variant cells, the substitutions were mainly transversions (58% in S, 73% in G1). In the normal cells irradiated in S, the majority of the substitutions were G.C --> A.T, and most involved CC photoproducts in the transcribed strand. In the variant cells irradiated in S, substitutions involving cytosine in the transcribed strand were G.C --> T.A transversions exclusively. G.C --> A.T transitions made up a much smaller fraction of the substitutions than in normal cells (P < 0.02), and all of them involved photoproducts located in the nontranscribed strand. The data strongly suggest that XP variant cells are much less likely than normal cells to incorporate either dAMP or dGMP opposite the pyrimidines involved in photoproducts. This would account for their significantly higher frequency of mutants and might explain their abnormal delay in replicating a UV-damaged template.

Base Sequence↗

PubMed1993

Mutations induced by 1-nitrosopyrene and related compounds during DNA replication in human cells and induction of homologous recombination by these compounds.

The transformation of normal human cells into cancer cells is a multistep process. Evidence suggests that a minimum of five independent steps (changes) are required for the development of certain kinds of human cancer, as well as for malignant transformation of human cells in culture. Mutations are one of the mechanisms involved in bringing about such changes. A single DNA base substitution mutation can activate an oncogene or inactivate a tumor suppressor gene. Because the action of tumor suppressor genes is to prevent cells from becoming malignant, the activity of both copies of such genes must be eliminated before suppression is lifted. Homologous mitotic recombination between a mutant tumor suppressor gene allele and its non-mutant allele is one mechanism for accomplishing this. The present study was designed to investigate the mechanisms by which certain carcinogenic compounds found in diesel exhaust particles and structurally-related N-substituted aryl carcinogens induce such base substitution mutations and homologous recombination events in mammalian cells in culture, including human cells. The system we employed to determine rapidly the kinds of mutations induced by these compounds, as well as the location of the point mutations in the target gene, involved a circular DNA molecule (plasmid) carrying a small target gene, supF. The target gene was exposed in vitro to radiolabeled compounds and then was allowed to replicate in human cells where the mutations were formed. The sites of mutation induction were compared with the sites of stable binding of the carcinogens to the DNA (adducts). The system used to determine whether these agents could induce homologous recombination consisted of a thymidine kinase-deficient mouse L cell line with a recombination substrate stably integrated into the genome. To determine whether or not excision repair was involved in the mechanism by which carcinogens induced recombination, the recombination substrate was introduced into an excision repair-proficient human cell line and two repair-deficient human cell lines. These cell lines were then compared for the frequency of recombination induced by the agents. All four N-substituted aryl compounds tested in the supF mutagenesis assay produced mainly base substitutions involving guanosine-cytosine (G.C)* base pairs, primarily G.C-->thymidine-adenine (T.A) transversions. However, 1,6-dinitropyrene adducts, formed by exposing the plasmids to 1-nitro-6-nitrosopyrene in the presence of a reducing agent, also induced a significant proportion (17%) of single G.C base-pair deletions.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1992

Mammalian expression vectors with modulatable promoters and two multiple cloning sites.

To facilitate the use of a wide range of selectable markers in transfection studies with human cells, in conjunction with the use of modulatable promoters for regulated expression of the genes of interest, we constructed two pUC19-based mammalian expression vectors, each containing two lacZ alpha-based multiple cloning sites (MCS). Selectable markers can be inserted into the MCS derived from pUC19, and the recombinants can be screened by lacZ complementation. The genes of interest can be inserted into the second MCS. The new MCS contains an amber stop codon in-frame with translation of the LacZ alpha-peptide. The presence of insert in the second MCS can also be screened on XGal plates, but in an Escherichia coli host containing an amber suppressor gene. Expression of the genes of interest can be modulated through transcription from the promoter of the mouse metallothionein-I-encoding gene or the long terminal repeat of the mouse mammary tumor virus. These vectors, as well as several of the intermediate plasmids described in this report, can be used to clone any two genetic elements into a single plasmid.

Amino Acid Sequence↗

PubMed1992

Preferential repair and strand-specific repair of benzo[a]pyrene diol epoxide adducts in the HPRT gene of diploid human fibroblasts.

If excision repair-proficient human cells are allowed time for repair before onset of S phase, the premutagenic lesions formed by (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy- 7,8,9,10-tetrahydrobenzo[a]pyrene (benzo[a]pyrene diol epoxide, BPDE) are lost from the transcribed strand of the hypoxanthine (guanine) phosphoribosyltransferase (HPRT) gene faster than from the nontranscribed strand. No change in strand distribution is seen with repair-deficient cells. These results suggest strand-specific repair of BPDE-induced DNA damage in human cells. To test this, we measured the initial number of BPDE adducts formed in each strand of the actively transcribed HPRT gene and the rate of repair, using UvrABC excinuclease in conjunction with Southern hybridization and strand-specific probes. We also measured the rate of loss of BPDE adducts from the inactive 754 locus. The frequencies of adducts formed by exposure to BPDE (1.0 or 1.2 microM) in either strand of a 20-kilobase fragment that lies entirely within the transcription unit of the HPRT gene were similar; the frequency in the 14-kilobase 754 fragment was approximately 20% lower. The rates of repair in the two strands of the HPRT fragment differed significantly. Within 7 hr after treatment with 1.2 microM BPDE, 53% of the adducts had been removed from the transcribed strand, but only 26% from the nontranscribed strand; after 20 hr, these values were 87% and 58%, respectively. In contrast, only approximately 14% of the BPDE adducts were lost from the 754 locus in 20 hr, a value even lower than the rate of loss from the overall genome (i.e., 38%). These results demonstrate strand-specific and preferential repair of BPDE adducts in human cells. They suggest that the heterogeneous repair of BPDE adducts in the human genome cannot be accounted for merely by the greatly increased rate of the repair specific to the transcribed strand of the active genes, and they point to a role for the chromatin structure.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed1992

Malignant transformation of human fibroblast cell strain MSU-1.1 by (+-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo [a]pyrene.

Treatment of MSU-1.1 cells, a near-diploid, karyotypically stable, infinite life-span human fibroblast strain, with (+-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene induced focus formation. Eight independent foci were isolated and the cell strains developed from them were examined for characteristics of malignant cells. Each grew to a higher density in medium containing 1% serum than did the MSU-1.1 cells. Three of the eight grew rapidly in serum-free medium without added growth factors, formed colonies in agarose with diameters of greater than or equal to 120 microns at a frequency of 5-19%, exhibited loss of genetic material, and, when injected into athymic mice, formed sarcomas that reached 6 mm in diameter within 2-3 wk. One produced high-grade sarcomas (progressively growing, invasive tumors exhibiting high mitotic activity); the other two produced low-grade sarcomas (tumors with a lower degree of mitotic activity) that developed focal areas of high-grade malignant cells if left in the animals for greater than 4 wk. A fourth cell strain formed high-grade sarcomas only after 2.5-3 mo, but the tumor-derived cells analyzed showed the same growth properties as the three malignant cell strains described above, exhibited loss of genetic material, and, when reinjected into athymic mice, produced high-grade sarcomas with a short latency period.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed1992

Quantitative two-dimensional gel electrophoresis analysis of human fibroblasts transformed by ras oncogenes.

Quantitative two-dimensional gel electrophoresis was used to compare the cellular protein patterns of a normal foreskin-derived human fibroblasts cell line (LG1) and three immortal derivatives of LG1. One derivative, designated MSU-1.1 VO, was selected for its ability to grow in the absence of serum and is non-tumorigenic in athymic mice. The other two strains were selected for focus-formation following transfection with either Ha-ras or N-ras oncogenes and form high grade malignant tumors. Correspondence and cluster analysis provided a nonbiased estimate of the relative similarity of the different two-dimensional patterns. These techniques separated the gel patterns into three distinct classes: LG1, MSU-1.1 VO, and the ras transformed cell strains. The MSU-1.1 VO cells were more closely related to the parental LG1 than to the ras-transformed cells. The differences between the three classes were primarily quantitative in nature: 16% of the spots demonstrated statistically significant changes (P < 0.01, T test, mean ratio of intensity > 2) in the rate of incorporation of radioactive amino acids. The patterns from the two ras-transformed cell strains were similar, and variations in the expression of proteins that occurred between the separate experiments obscured consistent differences between the Ha-ras and N-ras transformed cells. However, while only 9 out of 758 spots were classified as different (1%), correspondence analysis could consistently separate the two ras transformants. One of these spots was five times more intense in the Ha-ras transformed cells than the N-ras.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Transformation, Neoplastic↗

PubMed1991

Replication of acetylaminofluorene-adducted plasmids in human cells: spectrum of base substitutions and evidence of excision repair.

In rats fed the liver carcinogen 2-acetylaminofluorene (AAF), the two most abundant types of DNA adduct are N-(deoxyguanosin-8-yl)-2-acetylaminofluorene and its deacetylated derivative. When plasmids carrying AAF adducts replicate in bacteria, the predominant mutations are frameshifts, whereas with deacetylated (AF) adducts, they are mainly base substitutions, just as we found when plasmids carrying AF adducts replicated in human cells. We have investigated the frequency and spectrum of mutations induced when a shuttle vector carrying AAF adducts (85% bound to the C8 position of guanine, 15% to the N2 position) replicated in human cells. The frequency induced per initial AAF adduct was higher than with AF adducts, but the kinds of mutations were similar--i.e., 85% base substitutions, principally G.C----T.A transversions. There was good correlation between the "hot spots" for mutations and hot spots for AAF adduct formation, suggesting that mutational hot spots reflect preferential binding of the carcinogen to DNA. 32P-postlabeling analysis of the adducts before and after the DNA was transfected into the human cells showed that there was no deacetylation of AAF adducts and that 85% of both types of adducts were removed within 3.5 hr, most probably by excision repair.

2-Acetylaminofluorene↗

PubMed1991

Xeroderma pigmentosum variant cells are less likely than normal cells to incorporate dAMP opposite photoproducts during replication of UV-irradiated plasmids.

Xeroderma pigmentosum (XP) variant patients show the clinical characteristics of the disease, with increased frequencies of skin cancer, but their cells have a normal, or nearly normal, rate of nucleotide excision repair of UV-induced DNA damage and are only slightly more sensitive than normal cells to the cytotoxic effect of UV radiation. However, they are significantly more sensitive to its mutagenic effect. To examine the mechanisms responsible for this hypermutability, we transfected an XP variant cell line with a UV-irradiated (at 254 nm) shuttle vector carrying the supF gene as a target for mutations, allowed replication of the plasmid, determined the frequency and spectrum of mutations induced, and compared the results with those obtained previously when irradiated plasmids carrying the same target gene replicated in a normal cell line [Bredberg, A., Kraemer, K. H. & Seidman, M. M. (1986) Proc. Natl. Acad. Sci. USA 83, 8273-8277]. The frequency of mutants increased linearly with dose, but with a slope 5 times steeper than that seen with normal cells. Sequence analysis of the supF gene showed that 52 of 53 independent mutants generated in the XP variant cells contained base substitutions, with 62 of 64 of the substitutions involving a dipyrimidine. Twenty-eight percent of the mutations involved A.T base pairs, with the majority found at position 136, the middle of a run of three A.T base pairs. (In the normal cells, this value was only 11%.) If the rate of excision of lesions from supF in the two cell lines is equal, our data suggest that XP variant cells are less likely than normal cells to incorporate dAMP opposite bases involved in photo-products. If such incorporation also occurs during replication of chromosomal DNA, this could account for the hypermutability of XP variant cells with UV irradiation.

Bacterial Proteins↗

PubMed1991

Lack of a cell cycle-dependent strand bias for mutations induced in the HPRT gene by (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene in excision repair-deficient human cells.

We showed previously that in repair-proficient human cells the location of the premutagenic lesion induced by (+-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10- tetrahydrobenzo(a)pyrene (BPDE), namely, the guanine in a G.C base substitution, in mutants derived from cells treated at the beginning of S phase just when the hypoxanthine (guanine) phosphoribosyltransferase gene is replicated, differs significantly from their location in cells treated 12 h prior to the beginning of S phase (early G1 phase) (R-H. Chen et al., Proc. Natl. Acad. Sci. USA, 87:8680-8684, 1990). This suggests that the cells preferentially remove BPDE adducts from the transcribed strand. We have now determined the kinds and location of independent mutations induced by BPDE in the coding region of the hypoxanthine (guanine) phosphoribosyltransferase gene of synchronized repair-deficient xeroderma pigmentosum cells (XP12BE, complementation group A), treated at S or in G1. Nineteen of 25 mutants derived from S-treated cells and 23 of 28 mutants from G1-treated cells contained base substitutions. Eighty-nine percent of these involved a G.C base pair, primarily G.C----T.A transversions. This is similar to the kinds of mutations we saw in the repair-proficient cells. However, in contrast to our earlier results, there was no change in strand distribution of premutagenic BPDE lesions. In both populations, approximately 26% of the base substitutions involving G.C base pairs had the G located in the transcribed strand, 5 of 18 in the S phase mutants, and 5 of 21 in the G1 phase mutants. These results support the hypothesis that the strong strand bias of induced mutations observed in the repair-proficient cells results from preferential repair of BPDE-induced DNA damage from the transcribed strand.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed1991

Malignant transformation of human fibroblasts correlates with increased activity of receptor-bound plasminogen activator.

To determine whether a relationship exists among urokinase plasminogen activator (u-PA) activity, tissue plasminogen activator (t-PA) activity, and the malignant transformation of human fibroblasts, we measured receptor-bound and secreted u-PAs and t-PA activity in fibroblast cell strains of a unique cell lineage and compared the results with the values obtained in human fibrosarcoma-derived cell lines and control cell lines. The lineage consists of four nonmalignant, infinite life span cell strains, clonally derived from a finite life span, neonatal foreskin-derived cell line or one of its derivatives and 10 malignant cell strains clonally derived from that same derivative. Seven of the latter were malignantly transformed by K-, H-, or N-ras oncogene transfection, two were obtained following carcinogen treatment, and one arose spontaneously. All 10 malignant strains in this lineage exhibited significantly higher levels of activity of receptor-bound u-PA than was found in the cell strain from which they arose or the nonmalignant cell strains derived from it. The ras oncogene-transformed malignant strains also exhibited significantly higher levels of activity of receptor-bound t-PA than their cell strain of origin. The other three malignant strains showed undetectable levels, consistent with their attaining the malignant state by an alternate process. The five fully malignant fibrosarcoma-derived cell lines tested also showed high levels of receptor-bound u-PA and t-PA. The majority (greater than or equal to 80%) of the nonmalignant control cell lines did not do so. The 10 malignant cell strains in the lineage also exhibited higher levels of activity of secreted high molecular weight u-PA or t-PA than did their cell strain of origin and the nonmalignant cell strains derived from it, as did the malignant fibrosarcoma-derived cell lines. The data suggest that the malignant state of human fibroblasts is always associated with high levels of activity of receptor-bound u-PA, and in addition cells transformed to the malignant state are very likely to exhibit high levels of receptor-bound t-PA and secreted forms of plasminogen activators.

Cell Transformation, Neoplastic↗

PubMed1991

Use of PCR amplification of cDNA to study mechanisms of human cell mutagenesis and malignant transformation.

PCR is widely employed to amplify short segments of genomic DNA to determine if a specific change has occurred. But some investigators need to sequence the entire coding region of mammalian genes to determine what specific changes have occurred. In 1989, we [Yang et al: Gene 83:347-354] described a method to copy mRNA of the hypoxanthine (guanine) phosphoribosyl transferase (HPRT) gene directly from the lysate of a clone of 6-thioguanine-resistant mutant diploid human fibroblasts without the need for RNA extraction or DNA template purification. To avoid detecting random changes introduced by polymerases, 100 to 500 cells from an individual clone, each containing the identical mutation, are lysed and the cDNA is amplified 10(10)-to 10(11)-fold to obtain 5 to 10 micrograms of DNA. The consensus sequence of the cDNA is determined by direct nucleotide sequencing. Using this method, we have investigated the kinds of mutations induced by carcinogens in the coding region of the HPRT gene and their location in the gene and examined the role of DNA repair in this process. Normal repair-proficient human cells and cells deficient in DNA repair were exposed to mutagens in exponential growth or synchronized and exposed at the beginning of S phase or in G1 phase several hr prior to DNA replication. The kinds and location of mutations in the HPRT gene were determined and knowledge of the nature of the DNA lesions formed by the various mutagens allowed assignment of the DNA strand in which the premutagenic lesion that gave rise to the mutation had been located. Related assays involving PCR have been used to determine the nature of mutations in the coding region of the H-, N-, or K-ras genes of tumor-derived malignant human cells and to determine whether or not such cells express specific growth factor genes.

Cell Transformation, Neoplastic↗