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J W Simons

Publications and source records attributed to J W Simons.

At least 73 records · Page 4Linked to original sources
PubMed1994

How variable is a spontaneous mutation rate in cultured mammalian cells?

The Luria-Delbrück fluctuation analysis provides a method to estimate mutation rates and is commonly applied in somatic cell genetics and in cancer biology. We developed an assay for a Luria-Delbrück fluctuation analysis using the mouse lymphoma cell line, GRSL13. As these cells grow in suspension, one can handle hundreds of parallel cultures using multiwell dishes and dispensers. This assay thereby allows not only an accurate determination of the mutation rate per cell generation but also makes it possible to determine at which time after seeding mutations take place. Using approx. 8000 parallel cultures it has been possible to test whether the mutation rate is constant during the assay. It has been found that the spontaneous mutation rate of GRSL13 cells decreases in the course of a fluctuation test from 2 x 10(-6) to about 2 x 10(-7)/cell/generation. It was shown that this increased replication fidelity may partly be caused by cell density: maintenance of cells at high cell density resulted in a spontaneous mutation rate of 0.7 +/- 4.0 x 10(-7) compared to 4.0 +/- 3.1 x 10(-7) for the standard protocol. In contrast, growing the cells at extremely low cell density resulted in an enhanced mutation rate of 7.7 +/- 1.3 x 10(-7). Thus altogether the mutation rate can vary from 2 x 10(-6) to 0.7 x 10(-7) (approx. 30-fold). These results show that the spontaneous mutation rate is not constant, but highly dependent on experimental conditions. As incomplete expression and metabolic cooperation cannot explain the findings, the data suggest that the fidelity of DNA replication is not fixed but open to variation. Hence, determination of replication infidelity in cultured cells needs rigorous standardization or/and application of controlled variation in culture conditions.

Animals↗
PubMed1994

The DCC gene: structural analysis and mutations in colorectal carcinomas.

DCC is a candidate tumor-suppressor gene encoding a protein with sequence similarity to cell adhesion molecules such as N-CAM. A set of overlapping YAC clones that contains the entire DCC coding region was isolated. Studies of this YAC contig showed that the DCC gene spans approximately 1.4 Mb. For elucidation of exon-intron structure, lambda phage clones containing all known coding sequences were isolated from a genomic library. These clones were used to demonstrate the existence of 29 DCC exons, and the sequences of the exon-intron boundaries were determined for each. Twenty-three polymorphic markers from chromosome 18 were then studied in a panel of primary colorectal tumors that had lost some, but not all, of chromosome 18. In most of these tumors, the region that was lost included DCC. Finally, Southern blot and PCR-based approaches were used to search for subtle mutations in several DCC exons. One tumor that had a point mutation in exon 28 was found, resulting in a proline to histidine substitution. A second tumor with a point mutation in intron 13 was also found. The regional map and genomic structure of DCC should provide the means to more extensively study DCC gene alterations and protein function in normal and neoplastic cells.

Amino Acid Sequence↗
PubMed1994

Inhibition of intercellular communication by 2,3,7,8-tetrachlorodibenzo-p-dioxin and dioxin-like PCBs in mouse hepatoma cells (Hepa1c1c7): involvement of the Ah receptor.

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) and the coplanar 3,3',4,4'-tetrachlorobiphenyl (3,3',4,4'-TCB) and 3,3',4,4',5,5'-hexachlorobiphenyl (3,3',4,4',5,5'-HCB) on intercellular communication (IC) were determined in order to investigate the in vitro tumor promoting potency of these compounds. 2,3,7,8-TCDD and the two coplanar PCBs tested caused a rapid (2 hr) and a sustained inhibition (48 hr) of IC in the mouse hepatoma cell line (Hepa1c1c7) to 20 and 50% of the unexposed control, respectively. Inhibition of IC was dose dependent with an EC50 range of about 50-100 pM for 2,3,7,8-TCDD, 2-5 nM for 3,3',4,4'-TCB, and 10-15 nM for 3,3',4,4',5,5'-HCB, respectively. A comparison of the IC inhibitory effects of 2,3,7,8-TCDD and PCBs with a well-known aryl hydrocarbon receptor (AhR)-mediated response, the induction of ethoxyresorufin O-deethylase (EROD) activity, in the same cells revealed EROD induction by 2,3,7,8-TCDD, 3,3',4,4'-TCB, and 3,3',4,4',5,5'-HCB with EC50 ranges of 100-200 pM, 20-70 nM, and 5-10 nM, respectively. The time course of IC inhibition was paralleled by EROD induction, although the time of onset of the response was earlier for IC (1 hr) than for EROD (2.5 hr). A role of the AhR in the inhibition of IC by 2,3,7,8-TCDD and PCBs was demonstrated by the lack of inhibition in AhR-defective Hepa1c1c7 cells. Transient inhibition of IC was observed in the mutant cells only at early time points (within 2 hr of exposure). These results and the observation that alpha-naphtoflavone (an AhR antagonist) greatly reduced the 2,3,7,8-TCDD-dependent sustained inhibition of IC strongly support a role of the AhR in the sustained inhibition of IC by these compounds. Furthermore, these data suggest that the mouse Hepa1c1c7 cells may be a good model in which to study in vitro tumor promoting capacity of dioxins, PCBs, and related compounds.

Animals↗
PubMed1994

Demonstration of a rational strategy for human prostate cancer gene therapy.

The potential efficacy and clinical feasibility of gene therapy for prostate cancer were tested. Efficacy was tested using the Dunning rat prostate carcinoma model. Rats with anaplastic, hormone refractory prostate cancer treated with irradiated prostate cancer cells genetically engineered to secrete human granulocyte-macrophage colony-stimulating factor (GM-CSF) showed longer disease-free survival compared to either untreated control rats or rats receiving prostate cancer cell vaccine mixed with soluble human GM-CSF. A gene modified prostate cancer cell vaccine thus provided effective therapy for anaplastic, hormone refractory prostate cancer in this animal model. An evaluation of the clinical feasibility of gene therapy for human prostate cancer based on these findings was then undertaken. Prostate cancer cells from patients with stage T2 prostate cancer undergoing radical prostatectomy were first transduced with MFG-lacZ, a retroviral vector carrying the beta-galactosidase reporter gene. Efficient gene transfer was achieved in each of 16 consecutive cases (median transduction efficiency 35%, range 12 to 65%). Cotransduction with a drug-selectable gene was not required to achieve high yield of genetically modified cells. Histopathology confirmed malignant origin of these cells and immunofluorescence analysis of cytokeratin 18 expression confirmed prostatic luminal-epithelial phenotype in each case tested. Cell yields (2.5 x 10(8) cells per gram of prostate cancer) were sufficient for potential entry into clinical trials. Autologous human prostate cancer vaccine cells were then transduced with MFG-GM-CSF, and significant human GM-CSF secretion was achieved in each of 10 consecutive cases. Sequential transductions increased GM-CSF secretion in each of 3 cases tested, demonstrating that increased gene dose can be used to escalate desired gene expression in individual patients. These studies show a preclinical basis for proceeding with clinical trials of gene therapy for human prostate cancer.

Gene Transfer Techniques↗
PubMed1994

The UV action spectra for the clone-forming ability of cultured human melanocytes and keratinocytes.

Melanocytes (skin type 2) and keratinocytes were irradiated with UV light of 254, 297, 302, 312 and 365 nm and the survival was measured. Clone-forming ability was chosen as the parameter for cell survival. Melanocytes were found to be less sensitive to UV light than keratinocytes (a difference of a factor 1.22-1.92 for the UV-C and UV-B wavelengths (254, 297, 301 and 312 nm) and a factor 6.71 for the UV-A wavelength (365 nm). Because melanin does not appear to protect against the induction of pyrimidine dimers the difference between melanocytes and keratinocytes in the UV-C and UV-B region could not be explained by the presence of melanin in the melanocytes. The relatively small difference can be explained by the longer cell cycle of melanocytes, which provides more time for the melanocytes to repair UV damage. In the UV-A region the difference between melanocytes and keratinocytes was much larger, suggesting that besides the longer cell cycle some additional factors must be involved in protection against UV-A light.

Cell Survival↗
PubMed1993

Immortalization of Syrian hamster embryo cells: probabilistic event or deterministic process.

Previous findings on the induction of immortalization in SHE cells have been explained with the activation/alteration hypothesis which postulates that treatment with a carcinogen results in the induction of a so-called "activated state" which enhances the rate of a probabilistic event in the progeny of the treated cells. This event is supposed to be a mutation. Because it has been recently indicated that in mammalian cells the switching on of signal transduction pathways by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or carcinogens can lead to genetic instability in the progeny of the treated cells, the possibility of an analogy between the induction of genetic instability and induction of immortalization after treatment with TPA was investigated. No effect of TPA was found on the rate of immortalization/cell/generation, not in otherwise untreated cells nor in cells treated with benzo(a)pyrene. TPA was found to enhance the life span of SHE cells. The life span of a culture correlated with its growth rate and its cell density at confluence both in the absence and presence of TPA. These correlations are supposed to reflect a regulation mechanism involved in the program of cellular senescence, and supposedly TPA can partly reverse this program. Treatment with benzo(a)pyrene also interferes with the life span resulting in premature senescence in most of the cells and extension of life span in a small fraction of the cells which subsequently can become immortal. Repeated switching from logarithmic growth to G0 also enhanced life span and rate of immortalization. The findings indicate that the activated state is a disturbance of a differentiation program affecting in SHE cells the program of cellular senescence and that, as an explanation for immortalization, epigenetic alterations causing a deterministic process of dedifferentiation in a subpopulation of the cells appear as plausible or perhaps even more plausible as a probabilistic mutation. This indicates that disturbance of differentiation might be among the causes of genetic instability.

Animals↗
PubMed1993

Nuclear morphometry accurately predicts recurrence in clinically localized renal cell carcinoma.

Despite careful clinical staging, as many as 30 percent of patients with pathologically, specimen-confined renal cell carcinoma (RCC) have unpredictable recurrence following surgery. Present pathologic and clinical staging systems cannot accurately predict those patients with high risk of disease recurrence from those who are cured by surgery alone. Advances in immunotherapy including gene therapy for RCC have dictated the need to identify RCC patients for adjuvant therapy protocols who have a high probability of recurrence following nephrectomy. Nuclear morphometric techniques developed at our institution have predicted prognosis for a variety of genitourinary tumors; it was used to predict recurrence among patients undergoing nephrectomy for localized RCC. This report is a retrospective study of 26 patients with RCC of similar age, stage (pT1-pT3), and grade. Fifteen were free of disease at a mean of 75.2 months, and 11 had distant disease recurrence at a mean of 27.1 months. Statistical analysis of a variety of nuclear shape descriptors accurately separated this group of patients based on disease recurrence. No nuclear shape descriptor predicted disease recurrence when nuclei within the region of the tumor with the highest grade were analyzed. However, the range of nuclear ellipticity (p = 0.007) best predicted disease recurrence when nuclei were selected in a random fashion. Multivariate analysis of the four best shape descriptors better predicted disease recurrence (p = 0.002) with a sensitivity of 73 percent and specificity of 100 percent. These results are encouraging and suggest that this technique might be used in identifying patients for adjuvant gene therapy.

Adult↗
PubMed1992

Immortalization of carcinogen-treated Syrian hamster embryo cells occurs indirectly via an induced process.

The hypothesis that rodent cells can be immortalized by the direct induction of a single mutation-like event was tested by initiating cultures of benzo(a)pyrene treated Syrian hamster embryo cells with low inocula and expanding these few cells maximally until senescence prevented further culturing or immortalization took place. According to the mutation hypothesis immortalization is hardly to be expected under these conditions. However, immortalization was frequently observed. Therefore the induction of immortalization appears indirect. The progeny of benzo(a)pyrene treated cells immortalized with a rate of 3.9 x 10(-8)/cell/generation, which is 64 times higher than the spontaneous rate. The results are in line with the probabilistic theory developed in 1980 by both Fernandez et al. (Proc. Natl. Acad. Sci. USA, 77: 7272-7276, 1980) and Kennedy et al. (Proc. Natl. Acad. Sci. USA, 77: 7262-7266, 1980), which states that treatment of cells with a carcinogen can result in a so-called activated state of the treated cells which is transmitted to the progeny and which results in an enhanced rate of transforming events.

Animals↗
PubMed1992

Induction of 6-thioguanine-resistant mutants in human diploid fibroblasts in vitro with ethylene oxide.

Human diploid fibroblasts (strain VH-10) were exposed to the direct-acting alkylating agent, ethylene oxide (EtO), in vitro, and the frequency of HPRT mutants was evaluated by selection in medium containing 6-thioguanine. A dose-dependent increase of the mutant frequency was found in the dose range of 2.5-10 mMh of EtO. The EtO-induced mutant frequency increased 5-19 times the background frequency at low or moderately toxic doses, which indicates that EtO is a strong mutagen in human fibroblasts in vitro. The mutagenic potency was 9.8 x 10(-6) per mMh.

Alkylating Agents↗
PubMed1992

Investigation of colon cancers for human papillomavirus genomic sequences by polymerase chain reaction.

Two previous studies have reported the presence of human papillomavirus (HPV) genomic sequences in colorectal cancers. We examined DNAs from 50 colorectal tumors for HPV sequences by polymerase chain reaction (PCR), using HPV consensus L1 primers for amplification. The PCR products were screened with a generic HPV probe consisting of amplimers of HPV-16 and HPV-18. All tumor DNAs were negative for HPV sequences. These results call into the question the reported association of colorectal cancers with HPVs.

Colonic Neoplasms↗
PubMed1992

Stress response induced by DNA damage leads to specific, delayed and untargeted mutations.

Cells of the mouse T-lymphoma line GRSL13 were treated with 8-methoxy-psoralen plus longwave ultraviolet light (PUVA) under conditions where the biological effects are mainly due to non-persistent DNA cross-links (PUVA-CL treatment). Fluctuation analysis showed that PUVA-CL treatment resulted in an enhancement of the mutation rate in the progeny of treated cells, which persisted until the eleventh generation after treatment. Since only 5 cross-links are available to account for 52 mutational events observed in the coding region, about 90% of the induced mutational events must have been untargeted. This was confirmed by molecular analysis of these mutations, which showed that 53% of the point mutations arose at sites which are not a target for psoralens. This supports the hypothesis that stress responses may give rise to untargeted mutagenesis. Further support for this hypothesis is provided by the observation that 8-methoxy-psoralen (8-MOP) or UVA alone (both of which are known to induce many pleiotropic effects) each acted as indirect mutagen by enhancing the mutation rate 2-4 fold in the progeny of treated cells.

Animals↗
PubMed1992

(6-4) photoproducts and not cyclobutane pyrimidine dimers are the main UV-induced mutagenic lesions in Chinese hamster cells.

A partial revertant (RH1-26) of the UV-sensitive Chinese hamster V79 cell mutant V-H1 (complementation group 2) was isolated and characterized. It was used to analyze the mutagenic potency of the 2 major UV-induced lesions, cyclobutane pyrimidine dimers and (6-4) photoproducts. Both V-H1 and RH1-26 did not repair pyrimidine dimers measured in the genome overall as well as in the active hprt gene. Repair of (6-4) photoproducts from the genome overall was slower in V-H1 than in wild-type V79 cells, but was restored to normal in RH1-26. Although V-H1 cells have a 7-fold enhanced mutagenicity, RH1-26 cells, despite the absence of pyrimidine dimer repair, have a slightly lower level of UV-induced mutagenesis than observed in wild-type V79 cells. The molecular nature of hprt mutations and the DNA-strand specificity were similar in V79 and RH1-26 cells but different from that of V-H1 cells. Since in RH1-26 as well as in V79 cells most hprt mutations were induced by lesions in the non-transcribed DNA strand, in contrast to the transcribed DNA strand in V-H1, the observed mutation-strand bias suggests that normally (6-4) photoproducts are preferentially repaired in the transcribed DNA strand. The dramatic influence of the impaired (6-4) photoproduct repair in V-H1 on UV-induced mutability and the molecular nature of hprt mutations indicate that the (6-4) photoproduct is the main UV-induced mutagenic lesion.

Animals↗
PubMed1992

A possible factor in genetic instability of cancer cells: stress-induced secreted proteins lead to decrease in replication fidelity.

Ultraviolet irradiation triggers a response in mammalian cells known as the UV response. Part of the UV response forms the enhanced synthesis of various extracellular proteins able to transmit the response to non-irradiated cells. Because several cancer-prone syndromes with enhanced genetic instability also have an enhanced synthesis of the same set of proteins without prior stimulation it is possible that induction of these stress responsive proteins may be involved in the process of carcinogenesis and genetic instability. To test this hypothesis mouse T lymphoma cells, GRSL13, were treated with the conditioned medium of UV-induced cells under various experimental conditions. Overall the mutation rate is enhanced 1.8-fold (P < 0.01). However, the degree of enhancement is strongly influenced by culture conditions. UV-induced factors only lead to an enhanced mutation rate when cells, both for the production and response to those factors, originate from a similar cell density. In addition, it was found that fresh medium interferes with this response. To eliminate the hindrance of these factors on the effect of the conditioned medium on the mutation rate, serum-starved cells at high density were treated with serum-free medium derived from high-density UV-irradiated cultures. Using these conditions a 2.8-fold (P < 0.002) enhancement of the mutation rate was found. Fluctuation analysis indicated that the enhancement is 10-fold during the first five generations after treatment. UV-induced factors have also been found to induce cell growth, and the degree of induction was linearly correlated with the enhancement in mutation rate. These experiments are in agreement with the hypothesis that induction of stress responses leads to genetic instability.

Animals↗
PubMed1992

A Chinese hamster ovary cell mutant (EM-C11) with sensitivity to simple alkylating agents and a very high level of sister chromatid exchanges.

We have isolated a Chinese hamster ovary cell mutant hypersensitive to monofunctional alkylating agents. The mutant, designed as EM-C11, showed hypersensitivity to ethyl methanesulfonate (EMS), methyl methanesulfonate and ethylnitrosourea (8-, 7- and 2-fold, respectively, based on D10 values). About 2-fold increased sensitivity towards 4-nitroquinoline-1-oxide and only slightly increased sensitivity to X-rays (1.4-fold) and mitomycin C treatment (1.6-fold) were found in this mutant. EM-C11 was not hypersensitive to UV irradiation nor to adriamycin. The EM-C11 cells showed approximately 10-fold higher level of spontaneous sister chromatid exchange. The level of spontaneous chromosomal aberrations was 2- to 3-fold higher, but the frequency of EMS-induced chromosomal aberrations was approximately 10-fold higher in the mutant cells, in agreement with the increased sensitivity to killing. As measured by alkaline elution, EM-C11 cells showed a defect in the rejoining of single-strand DNA breaks after exposure to X-rays and even more so after the EMS treatment. Genetic analysis revealed that the EM-C11 mutant belongs to the same complementation group as the EM9 mutant described earlier. The XRCC1 gene which complements the defect in EM9 also complements the defect in EM-C11, confirming that these two independently isolated mutants are defective in the same gene.

Alkylating Agents↗