Outcomes of extended radical resections for locally advanced and recurrent pelvic malignancy involving the aortoiliac axis.

AIM: Currently, there is no clear consensus on the role of extended pelvic resections for locally advanced or recurrent disease involving major vascular structures. The aims of this study were to report the outcomes of consecutive patients undergoing extended resections for pelvic malignancy involving the aortoiliac axis. METHODS: Prospective data were collected on patients having extended radical resections for locally advanced or recurrent pelvic malignancies, with aortoiliac axis involvement, requiring en bloc vascular resection and reconstruction, at a single institution between 2014 and 2018. RESULTS: Eleven patients were included (median age 60 years; range 31-69 years; seven women). The majority required resection of both arterial and venous systems (n = 8), and the technique for vascular reconstruction was either interposition grafts or femoral-femoral crossover grafts. The median operative time was 510 min (range 330-960 min). Clear resection margins (R0) were achieved in nine patients. The median length of stay was 25 days (range 7-83 days). Seven patients did not suffer an early complication. There was one serious complication (Clavien-Dindo ≥ 3), an arterial graft occlusion secondary to thrombus in the immediate postoperative period, requiring a return to theatre and thrombectomy. The median length of follow-up in this study was 22 months (range 4-58 months). CONCLUSION: This series demonstrates that en bloc major vascular resection and reconstruction can be performed safely and can achieve clear resection margins in selected patients with locally advanced or recurrent pelvic malignancy at specialist surgery centres.

Abdominoperineal excision in Australasia: clinical outcomes, predictive factors and recent trends of nonrestorative rectal cancer surgery.

AIM: The decision to perform an abdominoperineal excision (APR) rather than restorative bowel resection relies on a number of clinical factors. There remains great variability in APR rates internationally. The aim of this study was to demonstrate trends of APR surgery in low rectal cancer (< 6 cm from the anal verge) in Australasia and identify predictors of nonrestoration. METHOD: This study reviewed a prospectively maintained colorectal registry - the Binational Colorectal Cancer Audit (BCCA) - from general/colorectal surgical units across Australia and New Zealand. Data were analysed to determine factors predictive of nonrestorative resection. Patients were analysed based on the presence (control) or absence (comparison) of a primary anastomosis. RESULTS: Of 3628 patients with rectal cancer, 2096 were diagnosed with low rectal cancer between 2007 and 2017. The incidence of APR remained constant over the study period, with 58% of all resections of low rectal cancer being APR. The majority of resections were performed by consultants in urban hospitals (86% vs 14%). Tumours ≤ 3 cm from the anal verge, T4, M1 disease and neoadjuvant therapy were the greatest predictors of APR (P < 0.001). A significantly increased rate of restorative surgery was observed in public hospital settings (59% vs 41%, P < 0.05). The rate of positive circumferential resection margin (CRM) was 7.95%, with significantly increased rates in patients undergoing APR (12.2% vs 6.2%, P < 0.001). CRM positivity was increased in open approaches, T4, N2 and M1 staged disease and in an emergency/urgent setting (P < 0.001 and P < 0.045, respectively). Significantly increased wound and pulmonary complications were observed in the APR cohort (P < 0.01). CONCLUSION: The rates of APR in Australia and New Zealand remain high but are comparable to international figures, with one-third of rectal cancers being treated by APR. The main determinants of APR are tumour height, T stage and neoadjuvant therapy requirement. CRM positivity was higher in APR patients.

Malignancy and mesenteric panniculitis.

AIM: Mesenteric panniculitis (MP) is a chronic inflammatory process of the small bowel mesentery that has been reported in conjunction with malignancy. The objectives of the present study were to identify the frequency and type of cancers that may coexist with MP and whether these can be seen on the initial diagnostic computerised tomography (CT). METHOD: A prospective database was kept of patients diagnosed with MP in the Canterbury region of New Zealand between 1 January 2003 and 31 December 2014. CT scans were independently reviewed. Clinical records were reviewed and family doctors were contacted for additional information. RESULTS: There were 302 patients with possible MP identified and 259 in whom it was confirmed on review. Seventy-eight patients had a diagnosis of malignancy, with 54 having a current cancer (59 total cancers), 33 a past cancer and nine both. Of the 59 current cancers the most common primary sites were colorectum (19), lymph nodes (17), kidney (six) and prostate (four). Fifty-four were at sites included on an abdominal CT scan. At all sites [except prostate (0/4)] there were high rates of detection on CT with 44/54 cancers visible including 20/23 gastrointestinal tract, 14/17 lymphomas and 9/9 non-prostate urogenital tract malignancies. Six people were subsequently diagnosed with cancer after the index CT. CONCLUSION: When MP occurs in association with malignancy, the commonest primary sites are large bowel, the lymph nodes and the urogenital tract. In those with MP on imaging, any cancer except prostate can usually be seen on the index CT. Further extensive investigation in asymptomatic patients is therefore likely to be of low yield.

The effect of aging on the autophagic and heat shock response in human peripheral blood mononuclear cells.

Autophagy is a lysosome degradation pathway through which damaged organelles and macromolecules are degraded within the cell. A decrease in activity of the autophagic process has been linked to several age-associated pathologies, including triglyceride accumulation, mitochondrial dysfunction, muscle degeneration, and cardiac malfunction. Here, we examined the differences in the autophagic response using autophagy-inducer rapamycin (Rapa) in peripheral blood mononuclear cells (PBMCs) from young (21.8 ± 1.9 years) and old (64.0 ± 3.7 years) individuals. Furthermore, we tested the interplay between the heat shock response and autophagy systems. Our results showed a significant increase in LC3-II protein expression in response to Rapa treatment in young but not in old individuals. This was associated with a decreased response in MAP1LC3B mRNA levels, but not SQSTM1/p62. Furthermore, HSPA1A mRNA was upregulated only in young individuals, despite no differences in HSP70 protein expression. The combined findings suggest a suppressed autophagic response following Rapa treatment in older individuals.

Effect of nucleotide excision repair in human cells on intrachromosomal homologous recombination induced by UV and 1-nitrosopyrene.

To study the role of nucleotide excision repair in the induction of intrachromosomal homologous recombination in mammalian cells, we introduced a plasmid containing a substrate for recombination into three human cell lines that differ in their repair capacity and compared the frequency of recombination induced by UV radiation and by 1-nitrosopyrene. One strain had a normal capacity for nucleotide excision repair, the second exhibited an intermediate rate of repair, and the third, derived from a patient with xeroderma pigmentosum, had no ability to repair UV- or 1-nitrosopyrene-induced DNA damage. The endogenous thymidine kinase genes in these cell strains had been inactivated, and the cells contained an integrated copy of a plasmid carrying duplicated copies of the herpes simplex virus type 1 thymidine kinase (Htk) gene, each inactivated by an 8-base-pair XhoI site inserted at a unique site. A functional tk gene can only be generated by a productive recombination event between the two Htk genes. In all three stains, UV and 1-nitrosopyrene induced dose-dependent increases in the frequency of recombinants. However, the doses required to cause a specific increase in recombination in the repair-deficient strains were 10 to 30 times lower than the dose required for the cell strain with a normal capacity for repair. These results strongly suggest that unexcised DNA lesions, rather than excision repair per se, stimulate intrachromosomal homologous recombination. Southern blot analysis of DNA from representative recombinants indicated that in all cases one of the two Htk genes had become wild type (XhoI resistant). The majority (90%) retained the Htk duplication, consistent with nonreciprocal transfer of genetic information (gene conversion).

Kinds and spectrum of mutations induced by 1-nitrosopyrene adducts during plasmid replication in human cells.

1-Nitropyrene has been shown in bacterial assays to be the principal mutagenic agent in diesel emission particulates. It has also been shown to be mutagenic in human fibroblasts and carcinogenic in animals. To investigate the kinds of mutations induced by this carcinogen and compare them with those induced by a structurally related carcinogen, (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetra-hydrobenzo [a]pyrene (BPDE) (J.-L. Yang, V. M. Maher, and J. J. McCormick, Proc. Natl. Acad. Sci. USA 84:3787-3791, 1987), we treated a shuttle vector with tritiated 1-nitrosopyrene (1-NOP), a carcinogenic mutagenic intermediate metabolite of 1-nitropyrene which forms the same DNA adduct as the parent compound, and introduced the plasmids into a human embryonic kidney cell line, 293, for DNA replication to take place. The treated plasmid, pZ189, carrying a bacterial suppressor tRNA target gene, supF, was allowed 48 h to replicate in the human cells. Progeny plasmids were then rescued, purified, and introduced into bacteria carrying an amber mutation in the beta-galactosidase gene in order to detect those carrying mutations in the supF gene. The frequency of mutants increased in direct proportion to the number of DNA-1-NOP adducts formed per plasmid. At the highest level of adduct formation tested, the frequency of supF mutants was 26 times higher than the background frequency of 1.4 X 10(-4). DNA sequencing of 60 unequivocally independent mutant derived from 1-NOP-treated plasmids indicated that 80% contained a single base substitution, 5% had two base substitutions, 4% had small insertions or deletions (1 or 2 base pairs), and 11% showed a deletion or insertion of 4 or more base pairs. Sequence data from 25 supF mutants derived from untreated plasmids showed that 64% contained deletions of 4 or more base pairs. The majority (83%) of the base substitution in mutants from 1-NOP-treated plasmids were transversions, with 73% of these being G . C --> T . A. This is very similar to what we found previously in this system, using BPDE, but each carcinogen produced its own spectrum of mutations. Of the five hot spots for base substitution mutations produced in the supF gene with 1-NOP, two were the same as seen with BPDE-treated plasmids. However, the three other hot spots were cold spots for BPDE-treated plasmids. Conversely, four of the other five hot spots seen with BPDE-treated plasmids were cold spots for 1-NOP-treated plasmids. Comparison of the two carcinogens for the frequency of supF mutants induced per DNA adduct showed that 1-NOP-induced adducts were 3.8 times less than BPDE adducts. However, the 293 cell excised 1-NOP-induced adducts faster than BPDE adducts.

Kinds of mutations formed when a shuttle vector containing adducts of benzo[a]pyrene-7,8-diol-9,10-epoxide replicates in COS7 cells.

We have investigated the kinds of mutations induced when a shuttle vector containing covalently bound residues of the (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) replicates in the monkey kidney cell line COS7. The target for detecting mutations was the 200-base pair gene for a tyrosine suppressor tRNA (supF), inserted at the EcoRI site in shuttle vector p3AC (Sarkar et al., Mol. Cell. Biol. 4:2227-2230, 1984). When introduced by transformation, a functioning supF gene in progeny plasmid recovered from COS7 cells allows suppression of a lacZ amber mutation in the indicator Escherichia coli host. Treatment of p3AC with BPDE caused a linear increase in the number of BPDE residues bound per plasmid. Untreated plasmids and plasmids containing 6.6 BPDE residues were transfected into COS7 cells, and the progeny were assayed for mutations in the supF gene. The frequency of mutants generated during replication of the BPDE-treated plasmids was not higher than that from untreated plasmids, but the two populations differed markedly in the kinds of mutations they contained. Gel electrophoresis analysis of the size alterations of 77 mutant plasmids obtained with untreated DNA and 45 obtained with BPDE-treated DNA showed that the majority of the mutant progeny of untreated plasmids exhibited gross alterations, principally large deletions. In contrast, the majority of the mutants generated during replication of the BPDE-treated plasmids contained only minor alterations, principally point mutations. Sequence analysis of progeny of untreated plasmids containing putative point mutations showed insertions and deletions of bases and a broad spectrum of base substitutions; in those from BPDE-treated plasmids, all base substitutions involved guanosine . cystosine pairs.

Site-specific excision repair of 1-nitrosopyrene-induced DNA adducts at the nucleotide level in the HPRT gene of human fibroblasts: effect of adduct conformation on the pattern of site-specific repair.

Studies showing that different types of DNA adducts are repaired in human cells at different rates suggest that DNA adduct conformation is the major determinant of the rate of nucleotide excision repair. However, recent studies of repair of cyclobutane pyrimidine dimers or benzo[a]pyrene diol epoxide (BPDE)-induced adducts at the nucleotide level in DNA of normal human fibroblasts indicate that the rate of repair of the same adduct at different nucleotide positions can vary up to 10-fold, suggesting an important role for local DNA conformation. To see if site-specific DNA repair is a common phenomenon for bulky DNA adducts, we determined the rate of repair of 1-nitrosopyrene (1-NOP)-induced adducts in exon 3 of the hypoxanthine phosphoribosyltransferase gene at the nucleotide level using ligation-mediated PCR. To distinguish between the contributions of adduct conformation and local DNA conformation to the rate of repair, we compared the results obtained with 1-NOP with those we obtained previously using BPDE. The principal DNA adduct formed by either agent involves guanine. We found that rates of repair of 1-NOP-induced adducts also varied significantly at the nucleotide level, but the pattern of site-specific repair differed from that of BPDE-induced adducts at the same guanine positions in the same region of DNA. The average rate of excision repair of 1-NOP adducts in exon 3 was two to three times faster than that of BPDE adducts, but at particular nucleotides the rate was slower or faster than that of BPDE adducts or, in some cases, equal to that of BPDE adducts. These results indicate that the contribution of the local DNA conformation to the rate of repair at a particular nucleotide position depends upon the specific DNA adduct involved. However, the data also indicate that the conformation of the DNA adduct is not the only factor contributing to the rate of repair at different nucleotide positions. Instead, the rate of repair at a particular nucleotide position depends on the interaction between the specific adduct conformation and the local DNA conformation at that nucleotide.

Carcinogens can induce homologous recombination between duplicated chromosomal sequences in mouse L cells.

The ability of a series of DNA-damaging agents to induce homologous intrachromosomal recombination between duplicated genes in the chromosome of mouse cells was investigated. The target cells were the thymidine kinase-deficient mouse L-cell strain 333M, which contains a single integrated copy of a plasmid with two herpes simplex virus thymidine kinase (Htk) genes, each containing an 8-base-pair XhoI linker inserted at a unique site. Expression of a functional Htk enzyme requires a productive recombinational event between the two nonfunctional genes. The spontaneous rate of recombination in this strain is 3 per 10(6) cells per generation. The agents tested represent physical carcinogens (UV and ionizing radiation), a simple alkylating agent (N-methyl-N'-nitro-N-nitrosoguanidine), an alkylating cross-linking agent (mitomycin C), and a reactive metabolite of a polycyclic aromatic hydrocarbon ((+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene [BPDE] ). The background frequency of tk+ recombinants in the untreated population averaged 18 X 10(-6) +/- 5 X 10(-6). Ionizing radiation had little or no effect on recombination; exposure to mitomycin C, N-methyl-N'-nitro-N-nitrosoguanidine, BPDE, or UV, at doses that lowered the survival to between 90 and 10% of the control, caused a dose-dependent increase in frequency of recombinants, reaching 50 X 10(-6) to 100 X 10(-6). No tk+ cells could be generated with a control cell line that contained only one mutant copy of the Htk gene. Molecular hybridization analysis showed that 85 to 90% of the tk+ recombinants retained the Htk gene duplication, consistent with nonreciprocal transfer of wild-type genetic information, gene conversion. In the rest, only a single copy of the Htk gene remained, reflecting a single reciprocal exchange within a chromatid or a single unequal exchange between sister chromatids. Each recombinant tested contained an XhoI-resistant (wild-type) Htk gene.

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.

Abnormal, error-prone bypass of photoproducts by xeroderma pigmentosum variant cell extracts results in extreme strand bias for the kinds of mutations induced by UV light.

Xeroderma pigmentosum (XP) is a rare genetic disease characterized by a greatly increased susceptibility to sunlight-induced skin cancer. Cells from the majority of patients are defective in nucleotide excision repair. However, cells from one set of patients, XP variants, exhibit normal repair but are abnormally slow in replicating DNA containing UV photoproducts. The frequency of UV radiation-induced mutations in the XP variant cells is significantly higher than that in normal human cells. Furthermore, the kinds of UV-induced mutations differ very significantly from normal. Instead of transitions, mainly C-->T, 30% of the base substitutions consist of C-->A transversions, all arising from photoproducts located in one strand. Mutations involving cytosine in the other strand are almost all C-->T transitions. Forty-five percent of the substitutions involve thymine, and the majority are transversions. To test the hypothesis that the UV hypermutability and the abnormal spectrum of mutations result from abnormal bypass of photoproducts in DNA, we compared extracts from XP variant cells with those from HeLa cells and a fibroblast cell strain, MSU-1.2, for the ability to replicate a UV-irradiated form I M13 phage. The M13 template contains a simian virus 40 origin of replication located directly to the left or to the right of the target gene, lacZalpha, so that the template for the leading and lagging strands of DNA replication is defined. Reduction of replication to approximately 37% of the control value required only 1 photoproduct per template for XP variant cell extracts, but approximately 2.2 photoproducts for HeLa or MSU-1.2 cell extracts. The frequency of mutants induced was four times higher with XP variant cell extracts than with HeLa or MSU-1.2 cell extracts. With XP variant cell extracts, the proportion of C-->A transversions reached as high as 43% with either M13 template and arose from photoproducts located in the template for leading-strand synthesis; with HeLa or MSU-1.2 cell extracts, this value was only 5%, and these arose from photoproducts in either strand. With the XP variant extracts, 26% of the substitutions involved thymine, and virtually all were T-->A transversions. Sequence analysis of the coding region of the catalytic subunit of DNA polymerase delta in XP variant cell lines revealed two polymorphisms, but these do not account for the reduced bypass fidelity. Our data indicate that the UV hypermutability of XP variant cells results from reduced bypass fidelity and that unlike for normal cells, bypass of photoproducts involving cytosine in the template for the leading strand differs significantly from that of photoproducts in the lagging strand.

Cell cycle-dependent strand bias for UV-induced mutations in the transcribed strand of excision repair-proficient human fibroblasts but not in repair-deficient cells.

To study the effect of nucleotide excision repair on the spectrum of mutations induced in diploid human fibroblasts by UV light (wavelength, 254 nm), we synchronized repair-proficient cells and irradiated them when the HPRT gene was about to be replicated (early S phase) so that there would be no time for repair in that gene before replication, or in G1 phase 6 h prior to S, and determined the kinds and location of mutations in that gene. As a control, we also compared the spectra of mutations induced in synchronized populations of xeroderma pigmentosum cells (XP12BE cells, which are unable to excise UV-induced DNA damage). Among the 84 mutants sequenced, base substitutions predominated. Of the XP mutants from S or G1 and the repair-proficient mutants from S, approximately 62% were G.C----A.T. In the repair-proficient mutants from G1, 47% were. In mutants from the repair-proficient cells irradiated in S, 71% (10 of 14) of the premutagenic lesions were located in the transcribed strand; with mutants from such cells irradiated in G1, only 20% (3 of 15) were. In contrast, there was no statistically significant difference in the fraction of premutagenic lesions located in the transcribed strand of the XP12BE cells; approximately 75% (24 of 32) of the premutagenic lesions were located in that strand, i.e., 15 of 19 (79%) in the S-phase cells and 9 of 13 (69%) in the G1-phase cells. The switch in strand bias supports preferential nucleotide excision repair of UV-induced damage in the transcribed strand of the HPRT gene.

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.

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.

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.

Human cell-mediated benzo(a)pyrene cytotoxicity and mutagenicity in human diploid fibroblasts.

A human epithelial cell-mediated cytotoxicity and mutagenicity assay system for benzo(a)pyrene [B(a)P] has been developed with human fibroblasts as the target cells. Lethally X-irradiated human kidney carcinoma-derived epithelial cells, which had constant levels of B(a)P-metabolizing activity, were cocultivated with target human skin fibroblasts (XP12BE), which lack excision repair capability for B(a)P-DNA adducts. The optimal conditions determined for the cell-mediated cytotoxicity assay were a 48-hr exposure to B(a)P concentrations ranging from 0.1 to 1 microM at a metabolizing cell:target cell ratio of at least 1:1. Under these conditions, the frequency of mutations to 6-thioguanine resistance induced in the target XP12BE cells by B(a)P as well as the binding of tritium-labeled B(a)P to DNA was also shown to be concentration dependent. High-pressure liquid chromatography analysis of enzymatically degraded B(a)P-DNA adducts revealed two peaks: a major peak (82%) which cochromatographed with the guanosine adduct-standard synthesized from anti-isomeric-7,8-dihydrodiol-9,10-epoxide of B(a)P; and a minor peak (18%) which cochromatographed with the guanosine adduct-standard synthesized from syn-isomeric-7,8-diol-9,10-epoxide of B(a)P. Human liver carcinoma- and lung carcinoma-derived cell lines, capable of metabolizing B(a)P, proved equal to or better than the kidney carcinoma-derived cell line in producing cytotoxic B(a)P metabolites in the cell-mediated cytotoxicity assay with target XP12BE cells.

Abnormal sensitivity of human fibroblasts from xeroderma pigmentosum variants to transformation to anchorage independence by ultraviolet radiation.

One class of xeroderma pigmentosum (XP) patients, known as XP variants, inherit the characteristic predisposition to sunlight-induced skin cancer, but unlike the majority of XP patients, their cells do not exhibit a deficiency in rate of excision repair of ultraviolet (UV) radiation-induced DNA damage. XP variant cells are only slightly more sensitive than normal to killing by 254 nm UV radiation or simulated sunlight. But they are much more sensitive than normal to the induction of mutations by these agents. We investigated their sensitivity to UV-induced transformation to anchorage independence compared to that of normal cells. Low doses of UV (2 to 4.5 J/m2), doses which resulted in little or no measurable transformation in normal cells, caused a dose-dependent increase in the frequency of anchorage independent XP variant cells. Doses of 6 to 8 J/m3 were required to elicit a comparable response in the normal fibroblasts. Even when the two kinds of cells were compared at doses adjusted to give equal cytotoxicity, the frequency of transformation in the XP variant cells was higher than normal. Thus, their sensitivity to induction of anchorage independence by UV paralleled their sensitivity to UV-induced mutations.

Morphological transformation, focus formation, and anchorage independence induced in diploid human fibroblasts by expression of a transfected H-ras oncogene.

In an attempt to determine how normal human fibroblasts respond to high expression of the T24 H-ras oncogene, we tranfected such cells with the plasmid vector pHO6T1 (D. A. Spandidos and N. M. Wilkie, Nature (Lond.), 310:469-475, 1984), containing the T24 H-ras oncogene with 5' and 3' enhancer sequences, and the aminoglycoside phosphotransferase gene which confers resistance to the drug, G418. Approximately 1.5% of the G418-resistant colonies obtained after transfection and selection consisted of cells exhibiting obvious morphological transformation; i.e., they were highly refractile and more rounded than normal fibroblasts. DNA hybridization analysis showed that the morphologically transformed cells contained the transfected T24 H-ras oncogene, and radioimmunoprecipitation analysis showed that they were expressing the T24 H-ras protein product, M, 21,000 protein. Morphologically transformed cells formed colonies in soft agar at a frequency at least 60 times higher than that of cells that had been transfected with the control plasmid containing the normal cellular H-ras gene. Cells transfected with plasmid pHO6T1 could also be identified by their ability to form distinct foci when grown to confluence in nonselective medium following transfection. This study demonstrates that normal diploid human fibroblasts in culture can be transformed by transfection with a H-ras oncogene, and that such transformation correlates with expression of the mutant Mr 21,000 protein.

Malignant transformation of human fibroblast cell strain MSU-1.1 by N-methyl-N-nitrosourea: evidence of elimination of p53 by homologous recombination.

To determine whether N-methyl-N-nitrosourea (MNU) can induce malignant transformation of human fibroblasts and whether O6-methylguanine (O6-MeG) is involved, two populations of infinite life span cell strain MISU-1.1, differing only in level of O6-alkylguanine-DNA alkyltransferase, were treated with MNU and assayed for focus formation. MNU caused a dose-dependent increase in the frequency of foci in both groups, but the dose required was significantly lower in the cells lacking O6-alkylguanine-DNA alkyltransferase, indicating that O6-MeG was causally involved. Of 35 independent focus-derived strains assayed for p53 transactivating abilily, one was heterozygous, and 15 had lost all activity, 1 of 7 from untreated cells and 14 of 27 from MNU-treated cells. These results indicate that loss of p53 is not required for focus formation but may permit cells to form foci. Of 35 strains assayed for tumorigenicity, 10 formed malignant tumors with a short latency, all 10 lacked wild-type p53. The p53 heterozygous strain also formed tumors after a long latency, and the cells from those tumors lacked p53 transactivating ability. None of the 19 strains with wild-type p53 formed tumors. These results indicate that although loss of p53 is not sufficient for malignant transformation of MSU-1.1 cells, it may be necessary. Analysis of the p53 cDNA from several focus-derived strains lacking p53 activity revealed that each contained the same mutation, an A to G transition at codon 215, resulting in a change from serine to glycine. Because p53 can be inactivated by mutations at any one of a large number of sites, finding the same mutation in each strain assayed strongly suggests that the target population included a subpopulation of cells with this codon 215 mutation in one allele. Further analysis showed that all 15 focus-derived cells strains that lacked p53 transactivating activity contained two alleles, each with the same codon 215 mutation, and that the mutant allele in the heterozygous strain also had that mutatation. Analysis of the p arm of chromosome 17 of the focus-derived cell strains containing the codon 215 mutation revealed seven patterns of loss of heterozygosity, evidence of mitotic homologous recombination. Similar analysis of a separate series of cell strains, derived from foci induced by cobalt-60, revealed four patterns of loss of heterozygosity, only two of which had been found with those induced by MNU. These data suggest that homologous mitotic recombination, induced by O6-MeG in a subpopulation of cells heterozygous for p53 mutation, rendered the cells homozygous for loss of p53 activity, that this allowed the cells to form foci, and that although loss of p53 is not sufficient for malignant transformation, it predisposes cells to acquire the additional changes needed for such transformation.

Relationship between sensitivity of cells from patients with hereditary cutaneous malignant melanoma to killing and mutations by 4-nitroquinoline 1-oxide and adduct formation.

Hereditary cutaneous malignant melanoma (HCMM) is an autosomal dominant disease and develops mostly in patients with the hereditary dysplastic nevus syndrome (DNS). A previous study from this laboratory showed that fibroblasts derived from skin biopsies of four HCMM/DNS patients and one DNS patient were significantly more sensitive to the cytotoxic and mutagenic effects of 4-nitroquinoline 1-oxide (4-NQO) than three fibroblast cell lines from normal newborn males and one from a 45-yr-old normal donor. The survival curves of the HCMM/DNS or DNS cell lines fell into two groups, one with a slope approximately twice as steep as the controls and one with a slope approximately 3 times as steep. This marked difference in survival curves disappeared if the same cell lines were exposed to 4-hydroxyaminoquinoline 1-oxide, a partially reduced metabolite of the parent compound, suggesting that the difference in sensitivity reflected a difference in the cells' ability to metabolize the parent compound into a more reactive form. To see if such increased sensitivity to the cytotoxic effect of 4-NQO was an identifying characteristic of cells from HCMM/DNS and DNS patients and whether cells derived from unaffected members of such families were also more sensitive to 4-NQO than the controls, the survival curves of a series of 40 coded cell lines, derived from patients, family members, or normal donors of various ages, were compared in a blind study. The slopes of the survival curves of all the HCMM/DNS and DNS cell lines proved to be 2- to 3-fold steeper than those of the four previously studied control cell lines, bringing the total to nine of nine and eight of eight, respectively. Ten of the 28 coded cell lines from nonaffected family members or normal, age-matched donors proved to be as resistant as the four previously studied control cell lines and another foreskin-derived control cell line, bringing the total of relatively resistant cell lines to 15 of 33. However, the rest were just as sensitive as the cells from the patients. Mutagenesis studies showed that increased sensitivity to killing by 4-NQO was accompanied by increased sensitivity to mutation induction. Relatively sensitive and relatively resistant cell lines were treated with tritiated 4-NQO and assayed for survival and for the number of 4-NQO residues bound to DNA. The relationship between the number of adducts formed and cell survival was linear, with the relatively sensitive cell lines exhibiting the higher level of residues.(ABSTRACT TRUNCATED AT 400 WORDS)