Biomedical subjects

J L Redpath

Publications and source records attributed to J L Redpath.

At least 37 records · Page 2Linked to original sources

PubMed1998

Restored gap junctional communication in non-tumorigenic HeLa-normal human fibroblast hybrids.

Gap junctional intercellular communication (GJIC) has been implicated in homeostasis, development, differentiation, wound healing or regeneration and adaptive responses of differentiated cells. The dysfunction of homologous or heterologous GJIC has been associated with the tumorigenic phenotype. Restoration of growth control and the suppression of the tumorigenic phenotype have been previously associated with the up-regulation of GJIC by various anti-tumorigenic chemicals or transfection of connexin genes into tumor cells. To test the hypothesis that 'tumor suppressor' genes may be associated with the up-regulation of GJIC, we tested clones of tumorigenic HeLa, several non-tumorigenic HeLa-normal human fibroblast somatic cell hybrids and a tumorigenic segregant of one of the non-tumorigenic hybrids for GJIC. The parental HeLa cells (D98 AH.2) had no detectable GJIC but expressed detectable connexin 43 transcripts, while the non-tumorigenic HeLa-human fibroblast hybrids, which contained the chromosome 11 from the normal human fibroblast (CGL-1, CGL-2, ESH15 and EHS15c1), expressed ample connexin 43 transcripts and showed proficient GJIC. The tumorigenic segregant (CGL-3) from the non-tumorigenic HeLa-human fibroblast hybrid showed no GJIC or connexin 43. These results show that the presence of GJIC is closely linked to the suppression of the tumorigenic phenotype in the HeLa-human fibroblast hybrid and further suggest that GJIC may be associated with the mechanisms of tumor suppression. The mechanism by which the tumor suppressor gene(s) on the normal chromosome in the HeLa-human fibroblasts induces the up-regulation of connexin 43 is not yet explained.

Base Sequence↗

PubMed1998

Reduction of caveolin-1 expression in tumorigenic human cell hybrids.

Studies on human cell hybrids of a cervical carcinoma cell line, HeLa, and normal fibroblasts have indicated that the tumorigenicity of these cells is under the control of a putative tumor suppressor on chromosome 11, although the nature of this suppressor remains unknown. We examined the expression of caveolin-1, a protein component of caveolae of the plasma membrane in these cell hybrids. The non-tumorigenic cell hybrid, CGL1, and normal fibroblast WI38 cells expressed 21-24 kDa caveolin-1, whereas in tumorigenic hybrid CGL4 as well as in the parental HeLa cells, the level of caveolin-1 was markedly reduced. Caveolin-1 expression was also reduced in gamma-ray-induced tumorigenic clones (GIMs) isolated from CGL1 cells, whereas non-tumorigenic irradiated cells expressed the same level of caveolin-1 as CGL1 cells. In accordance with these changes, the cellular level of caveolin-1 mRNA was reduced in the tumorigenic CGL4 cells and GIMs without any detectable changes in the caveolin-1 gene. However, the in vivo tumor growth of CGL4 cells was not altered when caveolin-1 was stably overexpressed through the transfection of a human caveolin-1 cDNA. These results suggest that reduction of caveolin-1 expression is necessary but not sufficient for emergence of the tumorigenic phenotypes of HeLa cell hybrids. Possible roles of the putative tumor suppressor in the control of gene expression are also discussed.

Blotting, Northern↗

PubMed1998

Loss of suppressor loci on chromosomes 11 and 14 may be required for radiation-induced neoplastic transformation of HeLa x skin fibroblast human cell hybrids.

We have previously reported a linkage between radiation-induced damage to a putative tumor suppressor locus on fibroblast chromosome 11 and the re-expression of tumorigenicity in a hybrid cell line (HeLa x human skin fibroblast) used to study neoplastic transformation. Further investigation into the molecular basis of radiation-induced neoplastic transformation of the hybrid cell, CGL1, indicates that loss of fibroblast chromosome 11 appears to be necessary but not sufficient for neoplastic transformation. Previous analysis had suggested, though not clearly demonstrated, a possible role for loss of alleles on fibroblast chromosome 14 in the neoplastic transformation of the hybrid cells. Therefore, the status of chromosome 14 in the gamma-ray-induced, neoplastically transformed (GIM) hybrid cell lines and in nontumorigenic control (CON) hybrid cell lines isolated from irradiated populations has been investigated. Chromosome painting and molecular studies using restriction fragment length polymorphisms and tetranucleotide repeat polymorphism analysis were performed. As an additional control, the status of chromosome 12 was also examined. We report that five of the eight GIM cell lines have lost one complete copy of a fibroblast chromosome 14 while only one of the five CON cell lines has lost a complete copy of a fibroblast chromosome 14. No evidence of large-scale loss of chromosome 12 was detected in the GIM or CON cells. The data further suggest that both copies of fibroblast chromosome 14 contain an active tumor suppressor locus and that radiation-induced loss of either fibroblast chromosome 14 is associated with neoplastic transformation in this system. We now conclude that loss of alleles on both fibroblast chromosome 11 and 14 may be required for the radiation-induced neoplastic transformation of these human hybrid cells.

Alkaline Phosphatase↗

PubMed1998

Induction of an adaptive response against spontaneous neoplastic transformation in vitro by low-dose gamma radiation.

An adaptive response against spontaneous neoplastic transformation in vitro induced by low-dose gamma radiation has recently been reported using a clone of C3H 10T1/2 cells with a predisposition toward spontaneous transformation (Azzam et al., Radiat. Res. 146, 369-370, 1996). To test the generality of this observation, the HeLa x skin fibroblast human hybrid cell system was used to look for such an adaptive response using a similar experimental protocol. In the experimental protocol used, the frequency of neoplastic transformation of unirradiated cultures (Arm A) was compared to that of cultures which had been irradiated with 1 cGy of gamma radiation and either plated immediately (Arm B) or held for 24 h at 37 degrees C prior to plating (Arm C) for assay of neoplastic transformation. The pooled data from four separate experiments demonstrated a significantly reduced transformation frequency for Arm C compared to Arms A and B. This is indicative of an adaptive response in the case of postirradiation holding, in agreement with the earlier study using C3H 10T1/2 cells. However, with the exception of one experiment, the existence of the adaptive response was not clear on the basis of analysis of individual experiments. This points out the importance of sample size when looking at low-dose effects that are close in magnitude to the inherent variations in spontaneous transformation frequency.

Adaptation, Physiological↗

PubMed1997

Altered N-glycosylation of glucose transporter-1 associated with radiation-induced tumorigenesis of human cell hybrids.

Studies on human cell hybrids between a cervical carcinoma cell line, HeLa, and normal fibroblasts have indicated that their tumorigenicity is under the control of a putative tumor suppressor on chromosome 11. We have previously demonstrated that a tumorigenic cell hybrid CGL4 expresses a larger glucose transporter, GLUT1, due to altered glycosylation when compared to the nontumorigenic counterpart CGL1. In this study, we demonstrated this glycosylation change in GLUT1 in gamma-ray-induced tumorigenic mutants (GIMs) isolated from CGL1 cells as expressing a tumor-associated surface antigen, intestinal alkaline phosphatase. In contrast, GLUT1 in the gamma-irradiated nontumorigenic control cells (CONs) did not show this alteration. In accordance with this glycosylation change, affinity to 2-deoxyglucose in these GIM clones was increased by about twofold when compared to the nontumorigenic CONs. These results suggest a close correlation between the glycosylation change in GLUT1 with increased affinity to D-glucose and tumorigenicity of these human cell hybrids.

Alkaline Phosphatase↗

PubMed1997

Subadditive interaction of radiation and Taxol in vitro.

PURPOSE: To examine the dependency of Taxol-radiation interactions on the scheduling of the two agents. METHODS AND MATERIALS: The human laryngeal squamous cell carcinoma line SCC20 was used for this study. Cells were irradiated as subconfluent cultures using Cs-137 gamma rays at a dose rate of 1.75 Gy/min. Cultures were pretreated with Taxol (7.5 nM for 12 h, S.F. = 0.4) and then irradiated with graded doses followed by either immediate plating or holding for 6 h either in the absence or presence of 7.5 nM Taxol prior to plating for colony-forming ability. Experiments in which cells were irradiated and then exposed to 7.5 nM Taxol for both 12 and 18 h were also performed. Parallel-flow cytometric analyses of cell-cycle distribution of the various treated populations were carried out. RESULTS: The results indicate that pretreatment with Taxol induced a G2 block which was maintained during 6 h postirradiation holding either in the presence or absence of Taxol. No modification of radiosensitivity in the low-dose region was seen for cells treated with Taxol, irradiated, and plated immediately, with the resulting survival being compatible with an additive effect. However, for Taxol-pretreated cells held for 6 h postirradiation, either in the absence or presence of Taxol, the resulting survival reproducibly demonstrated a marked less than additive effect. This was particularly prominent for cells held in the presence of Taxol. Subsequent experiments in which Taxol was added to cells immediately postirradiation again demonstrated a less than additive effect of the two modalities. CONCLUSION: The results of this study are consistent with a dual mechanism of action involving Taxol-induced radiation resistance, possibly as a consequence of postirradiation holding in G2, and radiation-induced Taxol resistance through an as-yet-undefined mechanism.

Antineoplastic Agents, Phytogenic↗

PubMed1997

High and low dose rate irradiation have opposing effects on cytokine gene expression in human glioblastoma cell lines.

Effects of radiation on five cytokine expressing human glioblastoma cell lines were studied. In comparison to unirradiated controls, IL-1 beta and IL-6 mRNAs were generally reduced after low (LDR, 1.0 cGy/min) and very low (VLDR, 0.35 cGy/min) dose rate irradiation. In contrast, high (HDR, 200 cGy/min) and intermediate (IDR, 4.1 cGy/min) dose rates increased steady-state levels of IL-1 beta and IL-6 mRNAs. The surviving fraction was generally inversely proportional to the dose rate; however, these glioma cells were unusually susceptible to LDR. In the two cell lines tested, IDR was less cytotoxic than either HDR or LDR irradiation. Although cytokine gene expression had no clear effect on radiation survival in vitro, autologous cytokines could be important to radiation response in vivo by affecting immune response, tumour stroma, vasculature or surrounding tissues. Adjusting dose rates to account for inverse dose rate effects and altered gene expression may be a useful strategy in optimising radiation therapy of glioblastomas.

Blotting, Northern↗

PubMed1997

Alpha-particle-induced neoplastic transformation in synchronized hybrid cells of HeLa and human skin fibroblasts.

Survival and oncogenic transformation frequencies were determined through the cell cycle in hybrid cells (HeLa x human skin fibroblasts), exposed to 0.30 and 0.15 Gy 4.3 MeV (LET= 101 keV/microm) alpha-particles. The cells were synchronized by mitotic collection and irradiated at times ranging from 2 to 10 h after collection, corresponding to G1 and early S. At 0.30 Gy the highest value in the transformation frequency (1.6 +/- 0.3) x 10(-4) transformants/survivor, occurred 4 h after mitotic collection, corresponding to mid-G1 and was about twice as high as that for the asynchronous population (0.7 +/- 0.1) x 10(-4) transformants/survivor. A similar pattern was seen at 0.15 Gy albeit less marked. The results are similar to previous findings with C3H10T1/2 exposed to 0.30 Gy where (1.8 +/- 0.4) x 10(-4) and (0.8 +/- 0.4) x 10(-4) transformants/survivor were found in mid-G1 and in the asynchronous population respectively. The results of both these studies with 101 keV/microm alpha particles indicate that mid-G1 cells may be more sensitive than asynchronous cells by up to a factor of two. However, it is unlikely that such a factor is sufficient to represent the cell cycle 'hot spot' for transformation postulated to explain the inverse dose-rate effect.

Alpha Particles↗

PubMed1997

Schedule dependence of the interaction of radiation and Taxol in HeLa cells.

Previous studies have indicated that the nature of the interaction of radiation and Taxol may be dependent on the cell line, drug dose and treatment schedule. The present study represents a systematic attempt to examine the schedule dependence of the interaction of radiation and Taxol in HeLa cells. The protocol used was radiation treatment (7 Gy), followed by a variable interval (0-24 h), followed by exposure to Taxol (7.5 nM, 24 h), followed by plating for a colony-forming assay. Parallel samples were also taken for flow cytometric analysis of the distribution of cells in the phases of the cell cycle at the beginning and end of Taxol treatment. The results indicate that sub-additive, additive or supra-additive interaction can be seen depending on the interval between the radiation and Taxol treatments. Full radiation survival curves were determined for cells exposed to Taxol either immediately or 10 h after the completion of radiation treatment, corresponding to sub-additive and supra-additive interactions, respectively. An examination of the data revealed that maximum cell killing occurred when the percentage of cells in G1 phase was at a minimum at the time of addition of Taxol. Studies of Taxol-induced toxicity using cells synchronized in G1 phase with mimosine and then released and allowed to progress through the cell cycle confirmed this observation. The conclusion of this study is that prior radiation treatment can modify the effect of subsequent Taxol treatment through alterations in the distribution of cells in the phases of the cell cycle. This finding has important implications for the clinical scheduling of these two cancer treatment modalities.

Cell Cycle↗

PubMed1996

DNA damage and p53-mediated cell cycle arrest: a reevaluation.

Most mammalian cells exhibit transient delays in the G1 and G2 phases of the cell cycle after treatment with radiation or radiomimetic compounds. p53 is required for the arrest in G1, which provides time for DNA repair. Recently, a role of p53 in the G2/M transition has also been suggested. However, it has been reported that the presence of functional p53 does not always correlate with the induction of these checkpoints. To precisely assess the role of p53 in activating cell cycle checkpoints and in cell survival after radiation, we studied the response of two isogenic human fibrosarcoma cell lines differing in their p53 status (wild type or mutant). We found that when irradiated cells undergo a wild-type p53-dependent G1 arrest, they do not subsequently arrest in G2. Moreover, wild-type p53 cells irradiated past the G1 checkpoint arrest in G2 but do not delay in the subsequent G1 phase. Furthermore, in these cell lines, which do not undergo radiation-induced apoptosis, the wild-type p53 cell line exhibited a greater radioresistance in terms of clonogenic survival. These results suggest that the two checkpoints may be interrelated, perhaps through a control system that determines, depending on the extent of the damage, whether the cell needs to arrest cell cycle progression at the subsequent checkpoint for further repair. p53 could be a crucial component of this control system.

Animals↗

PubMed1996

Reversion of UVC-induced tumorigenic human hybrid cells to the non-tumorigenic phenotype.

Non-tumorigenic HeLa x skin fibroblast human hybrid cells were UVC-irradiated (10 J/m2) and induced to neoplastic transformation with accompanying morphological change and expression of the HeLa tumour-associated antigen, intestinal alkaline phosphatase (IAP). A single-cell-derived cell line was cloned out of a neoplastically transformed focus and designated as UV-12. In low density culture, this cell line demonstrated the ability to undergo reversion to a morphology similar to that of the non-tumorigenic parent with accompanying, much reduced levels of IAP expression. The frequency of this reversion to low IAP expression increased with passage of low density cultures reaching 10(-2) at 26 passages. A revertant colony was selected and expanded into a cell line which was designated UV-12-RM-1. This cell line had a 67-fold reduction in IAP expression compared to UV-12 and demonstrated a much reduced tumorigenic phenotype. A cell line reconstituted from a tumour derived from this cell line demonstrated a high IAP expression level (3-fold less than UV-12) and was highly tumorigenic. Six single-cell-derived lines were cloned from UV-12-RM-1 and all had low IAP expression. Of these, one demonstrated an aggressive tumorigenicity, four showed the reduced tumorigenic phenotype characteristic of UV-12-RM-1, and one (UV-12-RM-105) was non-tumorigenic. However, with passage in culture, this latter cell line reverted to a weakly tumorigenic phenotype and a much elevated IAP level. It is hypothesised that the phenotypic shifts demonstrated by these UV-induced tumorigenic cells are under epigenetic control, and that they are most likely a consequence of an underlying genetic instability in the survivors of UVC-irradiation.

Alkaline Phosphatase↗

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↗

PubMed1994

The effect of postirradiation holding at 22 degrees C on the repair of sublethal, potentially lethal and potentially neoplastic transforming damage in gamma-irradiated HeLa x skin fibroblast human hybrid cells.

The effect of postirradiation holding at 22 degrees C on cell growth, progression of cells through the cell cycle, and the repair of sublethal, potentially lethal and potentially neoplastic transforming damage in gamma-irradiated HeLa x skin fibroblast human hybrid cells has been examined. Cell growth and cell cycle progression were essentially stopped at this reduced temperature. Cell survival was dramatically reduced by holding confluent cultures for 6 h at 22 degrees C, as opposed to 37 degrees C, after 7.5 Gy gamma radiation delivered at a rate of 2 Gy/min. Return of the cells to 37 degrees C for 6 h after holding at 22 degrees C did not result in increased survival. A similar effect was obtained when the cells were held at 22 degrees C between split-dose irradiation of log-phase cultures where no increase in survival was observed over a split-dose interval of 4 h. In this case a partial increase in survival was observed upon returning the cells to 37 degrees C for 3 h after holding at 22 degrees C for the first 3 h of the split-dose interval. Neoplastic transformation frequency was not enhanced by holding confluent cultures for 6 h at 22 degrees C after 7.5 Gy gamma radiation. This is consistent with previous observations that misrepair of potentially neoplastic transforming damage already occurs at 37 degrees C. The overall results are interpreted in terms of the reduced temperature favoring misrepair, rather than inhibition of repair, of sublethal, potentially lethal and potentially transforming radiation damage.

Cell Cycle↗

PubMed1993

Delayed heritable damage and epigenetics in radiation-induced neoplastic transformation of human hybrid cells.

The HeLa x skin fibroblast human hybrid cell system has proven to be an excellent model system for quantitative studies of radiation-induced neoplastic transformation in vitro. A unique aspect of this system is the reexpression of a cell surface protein p75/150 with tumorigenicity. The identification of p75/150 as intestinal alkaline phosphatase (IAP) allowed for the recent development of a more simplified, rapid, and sensitive screening method than the previous p75/150 antibody-based staining procedure. The new method directly detects neoplastically transformed, IAP-expressing cells by staining with the alkaline phosphatase chromogenic substrate, Western Blue (WB). Earlier studies with the antibody-based immunoperoxidase assay indicated that, while no foci with tumor-associated antigen (p75-positive) were evident 15 days after irradiation, the number of foci rose quickly and leveled off between Day 19 and Day 23. This late appearance of the IAP-positive foci suggested that the neoplastic transformation process was not an immediate consequence of radiation damage. The mechanism underlying this observation was unknown. The possibility existed that very small foci and/or foci expressing a low level of IAP were being missed at earlier expression times. The increased sensitivity of the WB staining technique has allowed for the reinvestigation of the kinetics of induction of radiation-induced foci in this system. Experiments were performed where parallel groups of transformation flasks were stained at Days 7, 9, 11, 13, 15, 17, 19, and 21 days after irradiation. The data clearly indicate that the radiation induction of IAP-positive foci is indeed delayed in this system with the vast majority of the foci beginning to appear after Day 9 after irradiation. The delay is not the result of a lack of ability to detect small IAP-positive foci since foci with as few as 15 IAP-positive cells were discernible. We have reported previously that under identical experimental conditions both the establishment of plateau phase and the onset of the expression of lethal mutations also occur after Day 9. We therefore propose that radiation-induced neoplastic transformation of HeLa x skin fibroblast hybrid cells is a consequence of the delayed expression of heritable damage under epigenetic control with a resultant loss of tumor-suppressor function.

Alkaline Phosphatase↗