Skin Fibroblast Human Hybrid Cells Research Articles

Threshold-Type Dose Response for Induction of Neoplastic Transformation by 1 GeV/nucleon Iron Ions

Neoplastic transformation of HeLa x skin fibroblast human hybrid cells by doses of 1 GeV/nucleon iron ions in the range 1 cGy to 1 Gy to exposed cultures has been examined. The data indicate a threshold-type dose-response curve with no increase in transformation frequency until doses above 20 cGy. At doses <10 cGy, not all exposed cells receive a direct traversal of an iron-ion track core, but all exposed cells receive up to several mGy of low-LET radiation associated with the delta-ray penumbra. It is proposed that the threshold-type response seen is a consequence of an adaptive response associated with the delta-ray exposure. For comparison purposes, the dose response for (137)Cs gamma rays over the same dose range was examined using the same experimental procedure. As we have shown previously, the dose response for (137)Cs gamma radiation was J-shaped. The iron ions were 1.5 to 1.7 times more biologically effective than the gamma radiation over the dose range examined.

Low Doses of Very Low-Dose-Rate Low-LET Radiation Suppress Radiation-Induced Neoplastic TransformationIn Vitroand Induce an Adaptive Response

The purpose of this study was to determine whether adaptation against neoplastic transformation could be induced by exposure to very low-dose-rate low-LET radiation. HeLa x skin fibroblast human hybrid cells were irradiated with approximately 30 kVp photons from an array of (125)I seeds. The initial dose rate was 4 mGy/day. Cell samples were taken at four intervals at various times over a period of 88 days and assayed for neoplastic transformation and the presence of reactive oxygen species (ROS). The dose rate at the end of this treatment period was 1.4 mGy/day. Transformation frequencies and ROS levels were compared to those of parallel unirradiated controls. At the end of 3 months and an accumulated dose of 216 mGy, cells treated with very low-dose-rate radiation were exposed to a high-dose-rate 3-Gy challenge dose of (137)Cs gamma rays, and the effects compared with the effect of 3 Gy on a parallel culture of previously unirradiated cells. Cells exposed to very low-dose-rate radiation exhibited a trend toward a reduction in neoplastic transformation frequency compared to the unirradiated controls. This reduction seemed to diminish with time, indicating that the dose rate, rather than accumulated dose, may be the more important factor in eliciting an adaptive response. This pattern was in general paralleled by a reduction of ROS present in the irradiated cultures compared to controls. The very low-dose-rate-treated cells were less sensitive to the high challenge dose than unirradiated controls, suggesting the induction of an adaptive response. Since there was a suggestion of a dose-rate threshold for induction suppression, a second experiment was run with a fresh batch of cells at an initial dose rate of 1 mGy/day. These cells were allowed to accumulate 40 mGy over 46 days (average dose rate=0.87 mGy/day), and there was no evidence for suppression of transformation frequency compared to parallel unirradiated controls. It is concluded that doses of less than 100 mGy delivered at very low dose rates in the range 1 to 4 mGy/day can induce an adaptive response against neoplastic transformation in vitro. When the dose rate drops below approximately 1 mGy/day, this suppression is apparently lost, suggesting a possible dose-rate-dependent threshold for this process.

Analysis of radiation effects using a combined cell cycle and multistage carcinogenesis model

Purpose To study radiation effects using a combined cell cycle and multistage clonal expansion model that includes processes of damage, repair, apoptosis, and mutation. The model includes endogenous and radiation induced damage causing progression of cells from normal, to damaged, to initiated, to initiated damage, to malignant status. We utilize complementary deterministic and stochastic versions of the model that share the same transition rates. The deterministic version is used to calibrate model rates for cell cycle progression, damage, checkpoint delay, repair, and apoptosis, and to implement tissue homeostasis. The stochastic version is used to predict the cancer hazard and survival. Results We calibrated transition rates in the deterministic version of the model to fit flow cytometry-based clonogenic survival data for Chinese hamster V79 cells and for HeLa × skin fibroblast human hybrid cells exposed to sparsely ionizing radiation during different phases of the cell cycle. We also calibrated repair and malignant transformation rates to fit neoplastic transformation data for HeLa × skin fibroblast human hybrid cells. We found that induced repair in G2 phase explained the low-dose hypersensitivity for survival in both cell lines, and a different induced repair process explained the neoplastic transformation data. Conclusion The shape of the induced repair curves for G2-phase survival and neoplastic transformation differ significantly, suggesting that these low-dose phenomena differ in regulation and, in fact, may be mechanistically unrelated.

Neoplastic transformation in vitro induced by low doses of 232 MeV protons

The aim was to define the dose – response curve for high-energy proton-induced neoplastic transformation in vitro. The HeLa × skin fibroblast human hybrid cell assay was used to determine the frequency of neoplastic transformation following doses of 232 MeV protons (mean linear energy transfer, LET = 0.44 keV μm – 1) in the range 5 – 600 mGy. Proton irradiations were carried out at the Loma Linda University Proton Treatment Facility, CA, USA. The data indicate no evidence for induction of transformation below a dose of 100 mGy. At doses of 5 and 50 mGy, there is evidence for a possible suppression of transformation frequencies below that for spontaneous transformation. The shape of the dose – response curve for high-energy proton-induced transformation of the human hybrid cell line CGL1 does not follow a linear no-threshold model and shows evidence for a threshold as well as for possible suppression of transformation at doses < 100 mGy, similar to that seen for other low-LET radiations.

Mechanisms of suppression of neoplastic transformation in vitro by low doses of low LET radiation.

Suppression of neoplastic transformation of HeLa x skin fibroblast human hybrid cells in vitro following low doses of low linear energy transfer radiation has been reported previously. The present study represents an exploration of two hypothesized mechanisms that may underlie this observed suppression. These are the up-regulation of reduced glutathione (GSH), a known antioxidant, and induction of DNA repair activity. The hybrid cells were found to have a high endogenous level of GSH and no induction following low doses of 60 kVp X-rays was observed. Buthionine sulfoximine (BSO), a GSH biosynthesis inhibitor, completely suppressed GSH levels in both unirradiated and irradiated cells. Furthermore, there was no significant impact of BSO-induced suppression of GSH on the neoplastic transformation frequency of either unirradiated or low dose irradiated cells indicating that glutathione levels play no role in the low dose suppression of transformation frequency. To assess the possible role of DNA repair in the low dose suppression of transformation the effect of 3-aminobenzamide (3-AB), a poly-ADP-ribose polymerase (PARP) inhibitor was examined. In these experiments, there was no significant effect of 3-AB on the transformation frequency at a dose of Cs-137 gamma rays of 0.5 cGy, however, at a dose of 5 cGy there was a significant increase (P < 0.05) in the transformation frequency in the presence of 3-AB. These findings suggest that the influence of DNA repair on the low dose suppression of transformation is significant at a dose of 5 cGy, but not at the lower dose of 0.5 cGy.

Low-dose reduction in transformation frequency compared to unirradiated controls: the role of hyper-radiosensitivity to cell death.

Calculations based on plausible parameters taken from the existing experimental database, and new measurements on the cell cycle dependence of low-dose hyper-radiosensitivity (HRS) of non-tumorigenic HeLa x skin fibroblast human hybrid cells, provide the first experimental evidence that the selective killing of a transformation-sensitive G(2)/M-phase subpopulation as a consequence of low-dose HRS could account in part for the observed reduction of induced transformation frequencies at low doses to values below that observed spontaneously. However, it is clear that other mechanisms associated with classical adaptive response, such as induced DNA repair, are also likely to be involved.

Low doses of diagnostic energy X-rays protect against neoplastic transformation in vitro

Purpose : To investigate the effect of low doses of 60 kVp X-rays on in vitro transformation frequency. Materials and methods : HeLa ×skin fibroblast human hybrid cells were used to assay transformation from the non-tumorigenic to the tumorigenic phenotype. Subconfluent cultures of cells were exposed to a range of doses of 60 kVp X-rays and seeded for assay of transformation after 24 h post-irradiation holding. Experiments were repeated at least three times and the data pooled for analysis. Transformation frequencies were compared with those of sham-irradiated controls. Results : At doses < 1 cGy, the observed transformation frequencies were significantly less than those seen in unirradiated cells. Conclusion : Low doses (< 1 cGy) of 60 kVp X-rays protect HeLa ×skin fibroblast human hybrid cells against neoplastic transformation in vitro.

The shape of the dose-response curve for radiation-induced neoplastic transformation in vitro: evidence for an adaptive response against neoplastic transformation at low doses of low-LET radiation.

A dose-response curve for gamma-radiation-induced neoplastic transformation of HeLa x skin fibroblast human hybrid cells over the dose range 0.1 cGy to 1 Gy is presented. In the experimental protocol used, the spontaneous (background) frequency of neoplastic transformation of sham-irradiated cultures was compared to that of cultures which had been irradiated with (137)Cs gamma radiation and either plated immediately or held for 24 h at 37 degrees C prior to plating, for assay for neoplastic transformation. The pooled data from a minimum of three repeat large-scale experiments at each dose demonstrated a reduced transformation frequency for the irradiated compared to the sham-irradiated cells for doses of 0.1, 0.5, 1, 5 and 10 cGy for the delayed-plating arm. The probability of this happening by chance is given by 1/2(n), where n is the number of observations (5); i.e., 1/32 congruent with 0.031. This is indicative of an adaptive response against spontaneous neoplastic transformation at least up to a dose of 10 cGy of gamma radiation. The high-dose data obtained at 30 and 50 cGy and 1 Gy showed a good fit to a linear extrapolation through the sham-irradiated, zero-dose control. The delayed-plating data at 10 cGy and below showed a statistically significant divergence from this linear extrapolation.

Kinetics of induction of reactive oxygen species during the post-irradiation expression of neoplastic transformation in vitro

Purpose : To examine whether the delayed expression of cell death and neoplastic foci in irradiated HeLa ×human skin fibroblast human hybrid cells correlates with the presence of reactive oxygen species (ROS) in the progeny of the irradiated cells. Material and methods : HeLa ×human skin fibroblast human hybrid cells were irradiated and plated as for assay of neoplastic transformation. At regular time intervals during the post-irradiation expression period of 21 days, samples were harvested and assessed for the presence of oxyradical activity by measuring the oxidation of dichlorohydrofluorescein (DCFH) to dichlorofluorescein (DCF) using flow cytometry. This reagent principally detects hydrogen peroxide. The kinetics of production of ROS were compared with those previously measured using the identical experimental protocol for the expression of both cell death and neoplastic foci. Results : The kinetics of production of ROS closely matched those for the onset of delayed cell death and delayed expression of neoplastic transformation. Conclusions : The induction of ROS is associated with delayed cell death and delayed neoplastic transformation in irradiated HeLa ×human skin fibroblast human hybrid cells.

Gap junction expression following UVC-induced neoplastic transformation in human hybrid cell lines.

Decreased connexin gene expression and loss of the capacity for either homologous or heterologous intercellular communication has been associated with neoplastic transformation. We tested the hypothesis that loss of gap junctional intercellular communication (GJIC) correlates with tumorigenic potential in the HeLa x skin fibroblast human hybrid cell system. Connexin gene expression, gap junction function and tumorigenicity were determined for the non-tumorigenic somatic hybrid cell line (CGL1) and a series of UVC-induced tumorigenic cell lines derived from CGL1. CGL1 and the parental skin fibroblasts express connexin43 (alpha1 gap junction gene) mRNA and protein, form gap junctional plaques and have functional gap junctions. UVC-irradiation of CGL1 cells produced a cell line (UV12) with an aggressive tumorigenic phenotype, which lost connexin43 expression as well as both homologous and heterologous GJIC and was in this respect similar to HeLa cells. However, the phenotype of UV12 cells exhibited some instability and revertants to a less aggressive tumorigenic phenotype were isolated. These cells expressed connexin43 mRNA and protein, and demonstrated homologous GJIC. Furthermore, cells reconstituted from a tumor derived from this revertant cell line retained significant connexin43 expression and homologous GJIC, although they exhibited an aggressive tumorigenic phenotype. Thus, functional homologous GJIC cannot be dissociated from tumorigenicity in this system. However, heterologous GJIC between these same UVC-induced tumorigenic cell lines and normal human skin fibroblasts was reduced, whereas the non-tumorigenic hybrid cells showed extensive heterologous GJIC. In summary, re-acquisition of connexin43 expression and homologous GJIC does not restore the non-tumorigenic phenotype in UVC-induced tumorigenic HeLa skin fibroblast human hybrid cells. However, reduction of heterologous GJIC does correlate with tumorigenicity in this cell system.

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.

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

HeLa 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 postirradiation 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.

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/m 2) 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 nontumorigenic parent with accompanying, much reduced levels of LAP expression. The frequency of this reversion to low LAP 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 LAP 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 LAP 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 LAP 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.

Reduced Temperature (22°C) Results in Enhancement of Cell Killing and Neoplastic Transformation in Noncycling HeLa × 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.

Late Mitosis/Early G 1 Phase and Mid-G 1 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).

The Effect of Postirradiation Holding at 22°C on the Repair of Sublethal, Potentially Lethal and Potentially Neoplastic Transforming Damage in Gamma-Irradiated HeLa × 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.

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.

A Simplified and Rapid Staining Method for the HeLa × 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.

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.