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J L Redpath

Publications and source records attributed to J L Redpath.

At least 19 recordsLinked to original sources
PubMed1995

Induction of cisplatinum sensitivity without alteration in radiation sensitivity by fractionated radiation treatment of a human laryngeal squamous cell carcinoma cell line.

PURPOSE: To determine if fractionated radiation treatment can alter cisplatinum sensitivity of a human laryngeal squamous carcinoma cell line. METHODS AND MATERIALS: Human squamous carcinoma cells, both previously untreated, as well as survivors of fractionated radiation therapy, were tested in vitro for their sensitivity to gamma radiation and cisplatinum. Fractionated gamma radiation was delivered in 14 or 10 daily fractions of 2 Gy. The cell line, cSCC-20, was derived from an untreated primary human laryngeal carcinoma. RESULTS: The human laryngeal squamous cell carcinoma cell line, cSCC-20, was demonstrated to have heterogeneous subpopulations with respect to cisplatinum sensitivity. No variation in radiation sensitivity was seen among subpopulations of varying cisplatinum sensitivity. The cells were relatively radioresistant (Do = 2.5 Gy). Fractionated radiation treatments of the parent cell line (14 fractions, 14 days, 2 Gy/fraction) or a cisplatinum sensitive subline (10 fractions, 12 days, 2 Gy/fraction) induced cisplatinum sensitivity (factor of 1.3 to 1.4) in the surviving cells. CONCLUSION: Fractionated radiation treatment of human squamous carcinoma cells in vitro induced sensitivity to cisplatinum without concomitant alteration in radiation sensitivity.

Carcinoma, Squamous Cell↗
PubMed1995

Late mitosis/early G1 phase and mid-G1 phase are not hypersensitive cell cycle phases for neoplastic transformation of HeLa x skin fibroblast human hybrid cells induced by fission-spectrum neutrons.

A two- to threefold increase in the rate of neoplastic transformation in cells irradiated at a dose rate of 0.22 cGy/min with fission-spectrum neutrons compared to that at 10.7 cGy/min has been confirmed with the use of alkaline phosphatase chromogenic substrate Western Blue staining to detect foci of neoplastically transformed cells through their expression of a tumor-associated antigen, the end point of the HeLa x skin fibroblast human hybrid cell transformation assay. To investigate whether the inverse dose-rate effect is due to the existence of a period in the cell cycle in which cells are significantly more sensitive to neoplastic transformation than in the rest of the cell cycle, as has been postulated previously (Rossi and Kellerer, Int. J. Radiat. Biol. 50, 353-361, 1986; Brenner and Hall, Int. J. Radiat. Biol. 58, 745-758, 1990; Elkind, Int. J. Radiat. Biol. 59, 1467-1475, 1991), we compared the sensitivity of late mitotic/early G1-phase and mid-G1-phase cells with that of asynchronous cells. The rationale for examining these particular cell cycle phases was based on the fact that mitosis has been hypothesized to be a candidate for the extremely sensitive period, and on a preliminary report that mid-G1-phase C3H 10T1/2 cells may exhibit enhanced sensitivity for neutron-induced transformation. A nominal dose of 45 cGy of fission-spectrum neutrons was delivered at approximately 10 cGy/min. The data indicate that neither late mitotic/early G1-phase nor mid-G1-phase cells are significantly more sensitive than asynchronous cells. Further, the dependence on the phase of the cell cycle for neoplastic transformation of CGL1 cells induced by fission-spectrum neutrons is different from that previously demonstrated for gamma radiation, where late-mitotic cells were approximately five times more sensitive than mid-G1-phase and asynchronous cells (Redpath and Sun, Radiat. Res. 121, 206-211, 1990).

Cell Cycle↗
PubMed1995

Reduced temperature (22 degrees C) results in enhancement of cell killing and neoplastic transformation in noncycling HeLa x skin fibroblast human hybrid cells irradiated with low-dose-rate gamma radiation.

The effect of reduced temperature (22 degrees C) or serum deprivation during low-dose-rate (0.66 cGy/min) gamma irradiation on cell killing and neoplastic transformation has been examined using the HeLa x skin fibroblast human hybrid cell system. The reduced temperature stops progression of these cells through the cell cycle while serum deprivation slows down cell turnover markedly. The data demonstrate an enhancement in both of the end points when cells are held at 22 degrees C compared to parallel experiments done at 37 degrees C. In operational terms, the decreased survival and increased neoplastic transformation are consistent with our earlier hypothesis of a higher probability of misrepair at reduced temperature (Redpath et al., Radiat. Res. 137, 323-329, 1994). The interpretation that this damage enhancement was associated with the reduced temperature, and not the fact that the cells were noncycling, was supported by the results of experiments performed with cells cultured at 37 degrees C in serum-free medium for 35 h prior to and then during the 12.24 h low-dose-rate radiation exposure. Under these conditions, cell cycle progression, as shown by reduction in growth rate and dual-parameter flow cytometric analysis, was considerably inhibited (cell cycle time increased from 20 h to 40 h), and there was no significant enhancement of cell killing or neoplastic transformation.

Cell Cycle↗
PubMed1995

Loss of a putative tumor suppressor locus after gamma-ray-induced neoplastic transformation of HeLa x skin fibroblast human cell hybrids.

The nontumorigenic HeLa x skin fibroblast hybrid cell line, CGL1, can be induced to re-express HeLa tumor-associated cell surface antigen, p75-IAP (intestinal alkaline phosphatase), with resulting neoplastic transformation, by exposure to gamma radiation. This has allowed the human hybrid system to be developed into a quantitative in vitro model for radiation-induced neoplastic transformation of human cells. Recently, several gamma-ray-induced IAP-expressing mutants (GIMs) of the nontumorigenic HeLa x skin fibroblast hybrid CGL1 were isolated and all were tumorigenic when injected subcutaneously into nude mice (Mendonca et al., Cancer Res. 51, 4455-4462, 1991). Control cell lines which were negative for p75-IAP (CONs) were also isolated from irradiated populations, and none were found to be tumorigenic. We have now begun to investigate the molecular basis of radiation-induced neoplastic transformation in this system by studying the potential genetic linkage between p75/IAP expression, tumorigenicity and damage to a putative tumor suppressor locus on fibroblast chromosome 11. Previous analysis of rare spontaneous segregants has indicated that this locus is involved in the regulation of tumorigenicity and in the expression of the HeLa tumor-associated cell surface marker intestinal alkaline phosphatase (p75-IAP) in this system. Therefore, analysis by restriction fragment length polymorphism and chromosome painting have been performed for chromosome 11, and for chromosome 13 as a control, for the p75/IAP-positive GIM and p75/IAP-negative CON cell lines. We report that in five of eight of the GIMs large-scale damage to the fibroblast chromosome 11's is evident (four GIMs have lost one complete copy of a fibroblast chromosome 11 and one GIM has both copies of fibroblast chromosome 11 heavily damaged). None of the CONs, however (0/5), have lost a complete copy of either fibroblast chromosome 11. No large-scale damage to the control chromosome 13's was detected in the GIMs or CONs. The data further suggest that both copies of fibroblast chromosome 11 contain an active locus and that radiation-induced loss of either fibroblast chromosome 11 will result in neoplastic transformation in this system. We conclude that it is the loss of a putative tumor suppressor locus on fibroblast chromosome 11 which is responsible at least in part for radiation-induced neoplastic transformation of these human hybrid cells.

Cell Transformation, Neoplastic↗
PubMed1994

Variable expression of IL-1 beta has minimal effect on the radiation sensitivity of three human glioma cell lines.

Interleukin 1 (IL-1) has been reported to act as a radioprotector in vivo. Data from our laboratory and from other investigators suggest that glioma cell lines can produce bioactive cytokines including IL-1 and also express IL-1 receptors. In view of the putative radioprotective effect of this cytokine, we have examined the in vitro radiosensitivity of three human glioma cell lines with widely varying levels of endogenous IL-1 beta. The data reveal that when irradiated (2 Gy/min) as confluent cultures (conditions optimal for differential IL-1 beta expression), and plated for colony formation after postirradiation holding, cell survival was not correlated with level of IL-1 beta mRNA expression in the two IL-1-expressing cell lines. However, this was not correlated with a further reduction in radiosensitivity. These data indicate that IL-1 beta does not act as an endogenous radioprotector in these cells under these experimental conditions.

Cell Count↗
PubMed1992

Lack of differential G2 chromosomal radiosensitivity between non-tumorigenic and tumorigenic human hybrid cells (HeLa x skin fibroblasts).

Previous studies have shown, that for log phase cultures, tumorigenic segregants of HeLa x skin fibroblast human hybrid cells are slightly more radiosensitive in terms of cell killing than their nontumorigenic parents (Redpath et al. 1985, Colman et al. 1988). Other studies have shown that these same tumorigenic segregants exhibit a markedly enhanced G2 chromosomal radiosensitivity (Sanford et al. 1986) thus offering a possible explanation for the cell killing data. The present study set out to examine the G2-phase radiosensitivity of these cells in terms of cell killing with the expectation that an enhanced sensitivity in the tumorigenic cells would be seen. No enhanced sensitivity was observed. The G2 chromosomal radiosensitivity was then examined and no differential was seen between the non-tumorigenic and tumorigenic cells. This lack of confirmation of previously reported studies may be due to some technical differences in the experimental protocols.

Cell Line, Transformed↗
PubMed1992

A simplified and rapid staining method for the HeLa x skin fibroblast human hybrid cell neoplastic transformation assay.

A simplified and rapid screening method for detecting radiation-induced neoplastically transformed foci in the HeLa x skin fibroblast human hybrid cell assay system has been developed. The method is based on the recent identification of the tumor-associated antigen in this system as intestinal alkaline phosphatase (IAP), and on the recent commercial development of a stable alkaline phosphatase chromogenic substrate solution, Western blue (WB). Cleavage of the substrate results in the production of a blue insoluble precipitate. It is shown that WB can be used on both viable and paraformaldehyde-fixed cells. Fixation does not noticeably reduce the IAP enzymatic activity. A direct comparison with the current method of immunoperoxidase (IMPO) staining indicates that the WB method is not only easier, but appears to be more sensitive in picking up weakly positive foci with a resulting higher (factor of 2.5) induced transformation frequency for 7 Gy of 137Cs gamma radiation. Whereas the IMPO staining procedure is time-consuming and requires access to large amounts of expensive IAP-specific BD6 monoclonal antibody and peroxidase-labeled secondary antibody, the WB staining procedure is rapid and utilizes an inexpensive and readily available reagent. It should now allow this assay system to enter general use.

Alkaline Phosphatase↗
PubMed1991

Characterization of intestinal alkaline phosphatase expression and the tumorigenic potential of gamma-irradiated HeLa x fibroblast cell hybrids.

Fusion of tumorigenic HeLa cells with human skin fibroblasts results in genetically stable hybrids which are nontumorigenic and no longer express the HeLa tumor-associated antigen, intestinal alkaline phosphatase (IAP). Previous analysis of spontaneous segregants of the nontumorigenic hybrid have implicated the loss of one copy of human fibroblast chromosome 11 with reexpression of IAP and tumorigenicity. This observation suggests that a putative HeLa tumor suppressor gene(s) is located on chromosome 11 and that this gene may be a negative regulator of the IAP gene. We have isolated several gamma-ray-induced mutants (GIMs) of the nontumorigenic HeLa x skin fibroblast hybrid CGL1 that were specifically selected for reexpression of IAP to further investigate the potential linkage between IAP regulation and the putative tumor suppressor locus. The GIMs have a wide range of cell morphology and level of IAP expression (nearly a factor of 40). The tumorigenicity of the GIMs was examined by s.c. injection into nude mice and all were found to be tumorigenic. The tumor volume-doubling time is in the range of 4 to 8 days for all the cell lines; however, the lag time to reach 500 mm3 tumor volume was significantly longer when the GIM IAP activity was low (less than 20% relative activity), suggesting perhaps that there is a threshold level of IAP expression required for tumor formation and selection for high IAP expression in vivo. However, studies with tumor reconstitutes of the GIMs and transfection studies with an IAP complementary DNA expression vector indicate that high IAP expression alone is not sufficient to confer rapid tumor growth. Therefore, while the data lend strong support to the continued tight correlation between IAP reexpression and tumorigenicity and to our proposal that the tumor suppressor may negatively regulate the IAP gene, it suggests that selection for other gene activities may be responsible for aggressive tumor growth in this cell hybrid system.

Alkaline Phosphatase↗
PubMed1991

The radiosensitivity of human keratinocytes: influence of activated c-H-ras oncogene expression and tumorigenicity.

We have investigated the gamma-ray sensitivity of several activated c-H-ras (EJ) containing clones that have been established after transfection of the spontaneously immortalized non-tumorigenic human keratinocyte cell line HaCaT. The clones were grouped according to their tumorigenic potential after subcutaneous injection into nude mice, and fell into three classes: Class I clones A-4 and I-6 are non-tumorigenic and express very low levels of c-H-ras mRNA and no mutated ras protein (p21); Class II clones I-5 and I-7 grow to large (benign) epidermal cysts, express intermediate to high c-H-ras mRNA and variable levels of mutated ras p21 protein with clone I-5 expressing little and clone I-7 expressing high levels of p21; Class III clones II-3 and II-4 grow to solid squamous cell carcinomas, express high c-H-ras mRNA and high level of mutated p21 ras protein similar to clone I-7. Comparison of the single-hit multitarget or linear-quadratic survival curve parameters, and survival at 2 Gy (S2) indicate that there appears to be no general correlation with either activated c-H-ras expression level or tumorigenic potential, and increased radioresistance.

Animals↗
PubMed1991

Effect of fission-neutron dose rate on the induction of a tumor-associated antigen in human cell hybrids (HeLa X skin fibroblasts).

The HeLa X skin fibroblast human cell hybrid system has been used to study the effect of fission-neutron dose rate on the induction of neoplastic transformation. Previously published data using neutrons from the JANUS reactor at Argonne National Laboratory are compared with some preliminary data obtained on the TRIGA reactor at the Armed Forces Radiobiology Research Institute. Comparable results were obtained on both reactors, both in terms of absolute values of transformation frequency and in terms of magnitude of the inverse dose-rate effect (a factor of 2.5 to 3).

Antigens, Neoplasm↗
PubMed1990

Suppression of radiation-induced neoplastic transformation of human cell hybrids by long term incubation at low extracellular pH.

We have previously reported that, when 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer was used in the growth medium to control pH fluctuations during the 21-day expression period of our human cell hybrid (HeLa x skin fibroblast) transformation assay, the yield of radiation-induced neoplastically transformed foci after 7 Gy of gamma-irradiation was suppressed. We now demonstrate that the observed suppression is not related to the presence of the 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer per se but rather is a function of the growth medium pH. Detailed studies reveal that incubation of the irradiated cells during the entire 21-day expression period at pH 6.7-6.8 versus pH 7.0-7.2 significantly suppressed the transformation frequency after 7 Gy, from 4.4 x 10(-4) to 4.6 x 10(-5) (accumulated data). The endpoint fraction of flasks containing foci was also significantly reduced at the lower pH. Suppression was evident whether the growth medium pH was lowered from pH 7.0-7.2 to pH 6.7-6.8 by medium exchange on day 0, 1, or 9 or even up to 15 days post-irradiation. Growth curves revealed that the population doubling time of the cells is extended and the unirradiated and irradiated plating efficiencies are lowered by long term low pH exposure. We discuss possible mechanisms for the observed suppression, in terms of the influence of low extracellular pH on cell turnover, repair of radiation damage, cell toxicity, and activity of cellular proteases.

Cell Division↗
PubMed1990

Fission-neutron-induced expression of a tumour-associated antigen in human cell hybrids (HeLa x skin fibroblasts): evidence for increased expression at low dose rate.

The induction of a tumour-associated antigen in a human cell hybrid line (HeLa x skin fibroblast) following exposure to fission neutrons of average energy 0.85 MeV (Janus reactor, Argonne National Laboratory) at two dose rates, 0.086 and 10.3 cGy/min, has been examined. The dose-response data obtained indicate the lower dose rate to be 2.9-fold more effective than the higher in inducing expression of the tumour-associated antigen, while there was no significant dose-rate effect in terms of cell killing. These results are qualitatively in agreement with previous observations using neutrons from the Janus reactor for the neoplastic transformation of C3H10T1/2 cells and Syrian hamster embryo cells.

Antigens, Neoplasm↗
PubMed1990

Sensitivity of a human hybrid cell line (HeLa X skin fibroblast) to radiation-induced neoplastic transformation in G2, M, and mid-G1 phases of the cell cycle.

The dependence of gamma-radiation-induced neoplastic transformation frequency on position in the cell cycle was measured for a human hybrid cell line (HeLa X skin fibroblast). The end point used was the induction of a tumor-associated antigen which in these cells correlates with tumorigenicity. Induction was measured in cells at G2, M, and mid-G1 phases and compared with the frequency induced in asynchronous cells. For studies of cells in G2 phase, the cells of an asynchronous population were collected for 3 h post-irradiation using the mitotic shake-off technique. For studies of cells in M and mid-G1 phases, cells were collected by mitotic harvest and then treated at the appropriate time. The data show that cells in G2 and M phase are very radiosensitive in terms of both cell killing and induction of neoplastic transformation compared to cells in mid-G1 or asynchronous populations. At a dose of 1 Gy, the transformation frequency was 10- to 20-fold higher for cells in M and G2 phase than for cells in mid-G1 or for asynchronous cells. However, the data indicate that the transformation frequencies were similar in the different phases of the cell cycle when correlated with surviving fraction. The results indicate that transformation frequency is more sensitive to changes in dose than is cell survival.

Cell Line↗
PubMed1989

Studies of the UVC-sensitivity of non-tumorigenic and tumorigenic human cell hybrids (HeLa x skin fibroblasts).

The UVC-sensitivities of a non-tumorigenic and a tumorigenic human cell hybrid (HeLa x skin fibroblasts) are compared and contrasted. The tumorigenic cells differ from the non-tumorigenic cells in that they have lost one copy each of chromosomes 11 and 14. For exponentially growing cultures, the tumorigenic cells are considerably more resistant than the non-tumorigenic cells. For confluent cultures, the differential in photosensitivity is much less. Flow cytometric studies of cell cycle distributions of both exponentially growing and confluent cultures of these cells indicate that the differences in photosensitivity cannot be explained by differences in cell cycle distribution. Studies of the kinetics of potentially lethal damage repair (PLDR) in confluent cultures of both cell lines indicate little or no recovery over the first 6 h followed by an increase in survival over the next 12-24 h. These data are consistent with previously published observations in human skin fibroblasts where the kinetics of PLDR reflected the kinetics of thymine dimer loss. The data are not consistent with 6-4 photoproducts being a potentially lethal lesion since such damage is rapidly repaired in human cells.

Cell Cycle↗
PubMed1989

UVC-induced expression of a tumor-associated antigen in human cell hybrids (HeLa X skin fibroblasts): repair of potentially lethal and potentially transforming damage.

The UVC-induced expression of a cell surface protein which correlates with the induction of tumorigenicity has been observed in human cell hybrids (HeLa X skin fibroblasts). A dose - response curve for the induction of this marker of neoplastic transformation has been obtained. Studies of the repair of potentially lethal and potentially transforming damage indicate a slow repair process which results in enhanced survival and decreased neoplastic transformation over the period 6-24 h post-treatment. The kinetics of this repair process are consistent with previously published work on the kinetics of removal of thymine dimers from UVC-irradiated human skin fibroblasts. During the first few hours post-irradiation (0-6 h) there are fluctuations in transformation frequency while survival remains essentially constant. It is during this period that 6-4 photoproducts are removed from UV-irradiated DNA. It is possible that repair of these lesions may lead to an increase in transformation frequency and have no impact in terms of survival.

Antigens, Neoplasm↗
PubMed1989

Plating efficiency as a function of time postirradiation: evidence for the delayed expression of lethal mutations.

The plating efficiency (PE) of a gamma-irradiated (7 Gy) human cell hybrid line (HeLa X skin fibroblast, designated as CGL1) has been measured as a function of time postirradiation and compared to that of unirradiated cells at similar cell densities and under the same growth conditions. The results indicate that following irradiation, the PE of the irradiated cells initially increases but never returns to that of unirradiated cells during the experimental period that we have examined. Furthermore, after a period of 9 to 10 days (equivalent to at least 10 cell doublings) postirradiation and plating, the PE of the irradiated cells begins to decrease and continues to do so over the next 5 days. A decrease does not occur in unirradiated cells until much later (i.e., Day 15) corresponding to at least 5 additional cell doublings. The data are discussed in terms of a delayed expression of lethal mutations. The possible impact of these observations on the estimation of radiation-induced transformation frequencies is also considered.

Cell Line↗