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M S Mendonca

Publications and source records attributed to M S Mendonca.

At least 19 recordsLinked to original sources
PubMed2001

Production of delayed death and neoplastic transformation in CGL1 cells by radiation-induced bystander effects.

Other investigators have demonstrated by transfer of medium from irradiated cells and by irradiation with low-fluence alpha particles or microbeams that cells do not have to be directly exposed to ionizing radiation to be detrimentally affected, i.e. bystander effects. In this study, we demonstrate by transfer of medium from X-irradiated human CGL1 hybrid cells that the killing of bystander cells reduces the plating efficiency of the nonirradiated CGL1 cells by 33 +/- 6%. In addition, we show that the amount of cell death induced by bystander effects is not dependent on X-ray dose, and that the induction of apoptosis does not appear to be responsible for the cell death. Furthermore, we found that the reduction in plating efficiency in bystander cells is evident for over 18 days, or 22 cell population doublings, after medium transfer, despite repeated refeeding of the cell cultures. Finally, we report the novel observation that bystander effects induced by the transfer of medium from irradiated cells can induce neoplastic transformation. Exposing unirradiated CGL1 cells to medium from cells irradiated with 5 or 7 Gy increased the frequency of neoplastic transformation significantly from 6.3 x 10(-6) in unirradiated controls to 2.3 x 10(-5) (a factor of nearly four). We conclude that the bystander effect induces persistent, long-term, transmissible changes in the progeny of CGL1 cells that result in delayed death and neoplastic transformation. The data suggest that neoplastic transformation in bystander cells may play a significant role in radiation-induced neoplastic transformation at lower doses of X rays.

Apoptosis↗
PubMed2000

The orphan receptor COUP-TFII regulates G2/M progression of breast cancer cells by modulating the expression/activity of p21(WAF1/CIP1), cyclin D1, and cdk2.

The orphan receptors COUP-TFI and COUP-TFII play an important role in development and differentiation by activating specific genes and by modulating the activity of nuclear receptors including estrogen receptor alpha (ERalpha) and retinoic acid receptors (RARs). Previously, it was demonstrated that the expression and activity of ERalpha and RARs are lost or impaired in anti-estrogen-resistant breast cancers. Here we show that, similar to ERalpha and RARs, the expression of COUP-TFII but not COUP-TFI is reduced in approximately 30% of breast cancer cell lines. Introduction of COUP-TFII to MDA-MB-435 cells resulted in reduced growth and plating efficiency. Interestingly, COUP-TFII increased the expression of cyclin D1 and p21(WAF1/CIP1) in MDA-MB-435 cells. Although parental and COUP-TFII-transduced cells progressed through the G1-S phase at a similar rate, progression of COUP-TFII cells through the G2/M transition phase was delayed. The activity of cdk2 required for G2/M progression was reduced in COUP-TFII cells compared to parental cells. This property of COUP-TFII is distinct from that of ERalpha and RARs, which usually modulate the G1 phase of breast cancer cells. Furthermore, these results reveal an important physiological function of COUP-TFII, which correlates with its ability to induce gene expression rather than modulation of nuclear receptor activity.

Breast Neoplasms↗
PubMed2000

Loss of chromosome 14 increases the radiosensitivity of CGL1 human hybrid cells but lowers their susceptibility to radiation-induced neoplastic transformation.

Loss of active tumor suppressor alleles on fibroblast chromosomes 11 and 14 are involved in radiation-induced neoplastic transformation of human hybrid CGL1 cells. Loss of either chromosome 11 or 14 alone is not sufficient for neoplastic transformation. To gain insight into the potential functions of these tumor suppressor loci, we have investigated the effects of chromosome 11 or 14 loss on radiation-induced neoplastic transformation. We recently demonstrated that loss of chromosome 11 increases the susceptibility to X-ray induced cell killing, neoplastic transformation and the expression of delayed death. The data suggested that one possible function of the chromosome 11 tumor suppressor gene may be to help maintain genome stability after radiation damage. We postulated that if the chromosome 14 allele is functioning in a similar manner, then the loss of chromosome 14 may also make the hybrid cells more susceptible to radiation-induced cell killing and neoplastic transformation. A hybrid cell line which has lost one copy of chromosome 14 was isolated and designated CON3(-14). CON3(-14) cells were more sensitive to X-ray-induced cell killing when compared with parental CGL1 cells. However, the susceptibility to radiation-induced neoplastic transformation was significantly reduced (by a factor of two) compared with the parental CGL1 cells. The expression of delayed death in the progeny of the irradiated CON3(-14) cells, growing in transformation flasks, was similar to CGL1 cells during the 21 day assay period. Taken together, the data indicate that loss of chromosome 14 alone increased the X-ray sensitivity of the hybrid cells but reduced their susceptibility to radiation-induced neoplastic transformation. These data suggest that the tumor suppressor alleles on chromosomes 11 and 14 may be functionally distinct in terms of their regulation of genomic instability and neoplastic transformation after radiation exposure.

Cell Survival↗
PubMed1999

Delayed apoptotic responses associated with radiation-induced neoplastic transformation of human hybrid cells.

HeLa X human skin fibroblast hybrid cells have been developed into a model for radiation-induced neoplastic transformation of human cells. Previous studies indicate that the appearance of neoplastically transformed foci in this system is delayed for several population doublings after irradiation and appears to involve the loss of putative tumor suppressor loci on fibroblast chromosomes 11 and 14. We now show that after treatment with 7 Gy of X-rays, transformed foci initiation correlates with delayed apoptosis initiated in the progeny of the irradiated cells after 10-12 cell divisions and with reduced plating efficiency (delayed death). The cells develop classic apoptotic morphology, positive terminal deoxynucleotidyl transferase-mediated nick end labeling and phosphatidylserine (annexin V) staining, and cleavage of poly(ADP-ribose) polymerase. In addition, a delayed induction of the p53 protein and the proapoptotic Bax protein is evident over a week after radiation exposure. We propose that a delayed build-up of mitosis-dependent genomic DNA damage or a loss of genetic material over time (10-12 cell divisions postirradiation) has two relevant outcomes: (a) cell death due to the delayed induction of a p53-dependent apoptosis; and (b) neoplastic transformation of a minor subset of survivors that has lost fibroblast chromosomes 11 and 14 (tumor suppressor loci for this system) and has either evaded apoptosis or not acquired enough genetic damage to induce apoptosis. It is postulated that both phenomena result from X-ray-induced, translesion-mediated genomic instability.

Apoptosis↗
PubMed1999

Previous loss of chromosome 11 containing a suppressor locus increases radiosensitivity, neoplastic transformation frequency and delayed death in HeLa x fibroblast human hybrid cells.

CGL1 (HeLa x fibroblast) hybrid cells have been utilized to study mechanisms of radiation-induced neoplastic transformation of human cells in vitro. Previous analysis has shown that loss of active tumor suppressor alleles on fibroblast chromosomes 11 and 14 may be required for radiation-induced neoplastic transformation of CGL1 cells. Loss of chromosome 11 alone was, therefore, found to be necessary but not sufficient for neoplastic transformation. We postulated that the loss of chromosome 11 may make the hybrid cells more susceptible to radiation-induced neoplastic transformation, since these cells have already undergone one of the required tumor suppressor loss events. Hybrid cells which have lost one copy of chromosome 11 were designated CON104(-11). CON104(-11) hybrid cells were found to have increased X-ray sensitivity and susceptibility to radiation-induced neoplastic transformation when compared with the parental CGL1 cells. In addition, the neoplastically transformed foci appear to arise earlier after radiation exposure in CON104(-11) versus CGL1 cells. Furthermore, the plating efficiency (PE) of the progeny of the irradiated CON104(-11) cells, growing in transformation flasks, is persistently lower than parental CGL1 cells during the 21 day assay period. The lower PE of the progeny of irradiated cells was attributed to the expression of delayed death/lethal mutations post-irradiation, a reflection of genomic instability. Taken together, the data indicate that previous loss of chromosome 11 may increase the radiation-induced genomic instability of the hybrid cells, leading to increased radiation sensitivity and neoplastic transformation potential. The data suggest that one possible function of the chromosome 11 tumor suppressor gene may be to help maintain genome stability after radiation damage.

Cell Death↗
PubMed1998

Evidence for a role of delayed death and genomic instability in radiation-induced neoplastic transformation of human hybrid cells.

HeLa x skin fibroblast human hybrid cells have been developed into a model of radiation-induced neoplastic transformation. The authors' studies indicate that the loss of putative tumour suppressor loci on fibroblast chromosomes 11 and 14 is evident after radiation-induced neoplastic transformation. How these fibroblast chromosomes/putative tumour suppressor loci are lost after radiation exposure is currently being investigated. It has been shown that the appearance of transformed foci correlates with the onset of the delayed reduction in plating efficiency or delayed death. This delayed death appears to be the result of the onset of a novel delayed apoptosis in the irradiated progeny beginning around day 8 post-irradiation. It was proposed that the reduction in plating efficiency and subsequent neoplastic transformation are all the result of a radiation-induced genomic instability. The instability process has two relevant outcomes: (1) cell death due to the induction of a delayed apoptosis in cells; and (2) neoplastic transformation of a small subset of survivors that have lost fibroblast chromosomes 11 and 14 (tumour suppressor loci) but either have not acquired enough genetic damage to induce the apoptotic response or have undergone molecular changes allowing them to bypass apoptosis. Data from the genomic instability and delayed death literature will be reviewed in terms of relevance to radiation-induced neoplastic transformation. New data are presented which demonstrate that use of growth media supplemented with a specific lot of calf serum was found to increase the number of cells undergoing radiation-induced neoplastic transformation, compared with standard serum after a fixed dose of radiation. This correlates with an increase in delayed death in the irradiated progeny which the authors propose is the result of increased genomic instability post-irradiation of cells grown in this serum. Preliminary data are presented indicating that a delayed apoptosis is also seen after high-energy He- particle exposure in this system.

Alpha Particles↗
PubMed1998

Molecular analyses of adaptive survival responses (ASRs): role of ASRs in radiotherapy.

Adaptive survival responses (ASRs), whereby cells demonstrate a survival advantage when exposed to very low doses of ionizing radiation (IR) 4 - 24 h prior to a high dose challenge, were first reported over 15 years ago. These responses were linked to hormesis, which implied that exposure to low levels of IR may be beneficial to the cell. We postulate that increased survival does not necessarily mean that the treatment is beneficial. Studies at the molecular level indicate that ASRs are the result of misregulated cell cycle checkpoint responses, occurring in the G1 phase of the cell cycle after IR. Specific gene products (i.e., PCNA, cyclin D1, cyclin A, XIP8, xip5 and xip13) appear to control these cell cycle checkpoint responses. Certain neoplastic cells show potent ASRs because they bypass checkpoints which would otherwise lead to apoptosis or other forms of cell death (possibly necrosis), and/or these cancer cells lack genetic factors, such as specific caspases (cysteine aspartate-specific proteases), that control apoptosis. Alterations in these cell cycle checkpoints or apoptotic responses may also occur during IR-induced stress responses in normal cells, at critical times (10-18 days posttreatment) following IR. One IR-induced protein, XIP8, may be a critical controlling factor at this point where delayed-onset apoptosis occurs. Additionally, we have shown that the presence or absence (i.e., SCID cells) of nonhomologous DNA double strand break repair did not seem to influence ASRs, suggesting that ASRs may be caused by signal transduction stress responses. ASRs may be beneficial to survival, however, the consequence(s) of that survival may be dire. For example, many neoplastic cells exhibited far greater ASRs than normal cells. Additionally, ASRs were induced by as little as 1 cGy and and were enhanced by repeated exposures of low level radiation. The implications for radiotherapy are that when a patient arrives for port film imaging during the course of therapy, the dose-rate, overall level of exposure, and time between port film exposure and high dose IR treatment become potentially important factors for improved efficacy of treatment of certain cancers. Further research is warranted to determine what molecular factors are most important for ASRs, and current work is focusing on XIP8.

Adaptation, Physiological↗
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↗
PubMed1997

In vitro cytotoxicity of silver-impregnated collagen cuffs designed to decrease infection in tunneled catheters.

PURPOSE: To examine in vitro the effects of silver-impregnated collagen cuff material from central venous catheters on human fibroblast growth. MATERIALS AND METHODS: In culture flasks, hybrid cells were exposed to silver-impregnated collagen cuff material, and human fibroblasts were exposed to silver-impregnated or silver-free collagen cuff material. After 72 hours of growth, cells were stained and digitally imaged, and the relative areas of cytotoxicity were determined. RESULTS: Flasks containing the silver-impregnated collagen cuff material and hybrid cells or human fibroblasts showed a marked local cytotoxic effect of the cuff material; cell-free zones surrounding the cuff material were demonstrated. No cytotoxic effect was seen in the flasks that contained silver-free cuff material (control group). Mean area of cleared cells was 312 mm2 +/- 130 (range, 156-624 mm2) in the flasks containing human fibroblasts and silver-impregnated cuff material and 0 mm2 in the corresponding control flasks (P < .0001). Mean radius of the area of cleared cells around the silver-impregnated cuff material in the flasks containing human fibroblasts was 9.8 mm +/- 2.0 (range, 7.0-14.1 mm). CONCLUSION: Silver-impregnated collagen cuff material demonstrates a local cytotoxicity on hybrid cells and human fibroblasts in vitro. This finding may explain the phenomena seen clinically of decreased anchorage and inadvertent removal of catheters with silver-impregnated collagen cuffs.

Animals↗
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

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↗
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↗
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

Differential repair of potentially lethal damage in exponentially growing and quiescent 9L cells.

The alteration of potentially lethal damage repair by postirradiation treatment with hypertonic saline (0.5 M PBS) was investigated in exponentially growing and quiescent 9L cells in vitro. A single dose of X rays (8.5 Gy) immediately followed by a 30-min treatment with hypertonic PBS at 37 degrees C reduced the survival of exponentially growing 9L cells by a factor of 13-18 compared to survival of irradiated immediately and delayed-plated cells, while the survival of quiescent cells was reduced by only a factor of 5-8. Survival curves confirmed the relative resistance of the quiescent 9L cells versus exponentially growing 9L cells to X rays plus hypertonic treatment. Both the slope and the shoulder of the survival curve were reduced to a greater extent in exponentially growing cells than in the quiescent cells by hypertonic treatment. The response of quiescent cells cannot be explained by either the duration of hypertonic treatment or the redistribution of the cells into G1 phase. We show that quiescent 9L cells can recover from hypertonically induced potentially lethal damage when incubated under conditions which have been found to delay progression through the cell cycle, and postulate that an altered chromatin structure or an enhanced repair capacity of quiescent 9L cells may be responsible for their resistance.

Animals↗