CGL1 Cells Research Articles

The Role of Natural Background Radiation in Maintaining Genomic Stability in the CGL1 Human Hybrid Model System.

Natural background ionizing radiation is present on the earth's surface; however, the biological role of this chronic low-dose-rate exposure remains unknown. The Researching the Effects of the Presence and Absence of Ionizing Radiation (REPAIR) project is examining the impacts of sub-natural background radiation exposure through experiments conducted 2 km underground in SNOLAB. The rock overburden combined with experiment-specific shielding provides a background radiation dose rate 30 times lower than on the surface. We hypothesize that natural background radiation is essential for life and maintains genomic stability and that prolonged exposure to sub-background environments will be detrimental to biological systems. To evaluate this, human hybrid CGL1 cells were continuously cultured in SNOLAB and our surface control laboratory for 16 weeks. Cells were assayed every 4 weeks for growth rate, alkaline phosphatase (ALP) activity (a marker of cellular transformation in the CGL1 system), and the expression of genes related to DNA damage and cell cycle regulation. A subset of cells was also exposed to a challenge radiation dose (0.1 to 8 Gy of X rays) and assayed for clonogenic survival and DNA double-strand break induction to examine if prolonged sub-background exposure alters the cellular response to high-dose irradiation. At each 4-week time point, sub-background radiation exposure did not significantly alter cell growth rates, survival, DNA damage, or gene expression. However, cells cultured in SNOLAB showed significantly higher ALP activity, a marker of carcinogenesis in these cells, which increased with longer exposure to the sub-background environment, indicative of neoplastic progression. Overall, these data suggest that sub-background radiation exposure does not impact growth, survival, or DNA damage in CGL1 cells but may lead to increased rates of neoplastic transformation, highlighting a potentially important role for natural background radiation in maintaining normal cellular function and genomic stability.

Overexpression of FRA1 (FOSL1) Leads to Global Transcriptional Perturbations, Reduced Cellular Adhesion and Altered Cell Cycle Progression.

FRA1 (FOSL1) is a transcription factor and a member of the activator protein-1 superfamily. FRA1 is expressed in most tissues at low levels, and its expression is robustly induced in response to extracellular signals, leading to downstream cellular processes. However, abnormal FRA1 overexpression has been reported in various pathological states, including tumor progression and inflammation. To date, the molecular effects of FRA1 overexpression are still not understood. Therefore, the aim of this study was to investigate the transcriptional and functional effects of FRA1 overexpression using the CGL1 human hybrid cell line. FRA1-overexpressing CGL1 cells were generated using stably integrated CRISPR-mediated transcriptional activation, resulting in a 2-3 fold increase in FRA1 mRNA and protein levels. RNA-sequencing identified 298 differentially expressed genes with FRA1 overexpression. Gene ontology analysis showed numerous molecular networks enriched with FRA1 overexpression, including transcription-factor binding, regulation of the extracellular matrix and adhesion, and a variety of signaling processes, including protein kinase activity and chemokine signaling. In addition, cell functional assays demonstrated reduced cell adherence to fibronectin and collagen with FRA1 overexpression and altered cell cycle progression. Taken together, this study unravels the transcriptional response mediated by FRA1 overexpression and establishes the role of FRA1 in adhesion and cell cycle progression.

Genomic Loss and Epigenetic Silencing of the FOSL1 Tumor Suppressor Gene in Radiation-induced Neoplastic Transformation of Human CGL1 Cells Alters the Tumorigenic Phenotype In Vitro and In Vivo.

The CGL1 human hybrid cell system has been utilized for many decades as an excellent cellular tool for investigating neoplastic transformation. Substantial work has been done previously implicating genetic factors related to chromosome 11 to the alteration of tumorigenic phenotype in CGL1 cells. This includes candidate tumor suppressor gene FOSL1, a member of the AP-1 transcription factor complex which encodes for protein FRA1. Here we present novel evidence supporting the role of FOSL1 in the suppression of tumorigenicity in segregants of the CGL1 system. Gamma-induced mutant (GIM) and control (CON) cells were isolated from 7 Gy gamma-irradiated CGL1s. Western, Southern and Northern blot analysis were utilized to assess FOSL1/FRA1 expression as well as methylation studies. GIMs were transfected to re-express FRA1 and in vivo tumorigenicity studies were conducted. Global transcriptomic microarray and RT-qPCR analysis were used to further characterize these unique cell segregants. GIMs were found to be tumorigenic in vivo when injected into nude mice whereas CON cells were not. GIMs show loss of Fosl/FRA1 expression as confirmed by Western blot. Southern and Northern blot analysis further reveals that FRA1 reduction in tumorigenic CGL1 segregants is likely due to transcriptional suppression. Results suggest that radiation-induced neoplastic transformation of CGL1 is in part due to silencing of the FOSL1 tumor suppressor gene promoter by methylation. The radiation-induced tumorigenic GIMs transfected to re-express FRA1 resulted in suppression of subcutaneous tumor growth in nude mice in vivo. Global microarray analysis and RT-qPCR validation elucidated several hundred differentially expressed genes. Downstream analysis reveals a significant number of altered pathways and enriched Gene Ontology terms genes related to cellular adhesion, proliferation, and migration. Together these findings provide strong evidence that FRA1 is a tumor suppressor gene deleted and epigenetically silenced after ionizing radiation-induced neoplastic transformation in the CGL1 human hybrid cell system.

A Preclinical Evaluation of Antrodia camphorata Alcohol Extracts in the Treatment of Non‐Small Cell Lung Cancer Using Non‐Invasive Molecular Imaging

This study was carried out to provide a platform for the pre-clinical evaluation of anti-cancer properties of a unique CAM (complementary and alternative medicine) agent, Antrodia camphorata alcohol extract (ACAE), in a mouse model with the advantageous non-invasive in vivo bioluminescence molecular imaging technology. In vitro analyses on the proliferation, migration/invasion, cell cycle and apoptosis were performed on ACAE-treated non-small cell lung cancer cells, H441GL and control CGL1 cells. In vivo, immune-deficient mice were inoculated subcutaneously with H441GL followed by oral gavages of ACAE. The effect of ACAE on tumor progression was monitored by non-invasive bioluminescence imaging. The proliferation and migration/invasion of H441GL cells were inhibited by ACAE in a dose-dependent manner. In addition, ACAE induced cell cycle arrest at G0/G1 phase and apoptosis in H441GL cells as shown by flow cytometric analysis, Annexin-V immunoflourescence and DNA fragmentation. In vivo bioluminescence imaging revealed that tumorigenesis was significantly retarded by oral treatment of ACAE in a dose-dependent fashion. Based on our experimental data, ACAE contains anti-cancer properties and could be considered as a potential CAM agent in future clinical evaluation.

The RBE of 3.4 MeV -particles and 0.565 MeV neutrons relative to 60Co -rays for neoplastic transformation of human hybrid cells and the impact of culture conditions

The neoplastic transformation of human hybrid CGL1 cells is affected by perturbations from external influences such as serum batch and concentration, the number of medium changes during the 21-day expression period and cell seeding density. Nevertheless, for doses up to 1.5 Gy, published transformation frequencies for low linear energy transfer (LET) radiations (gamma-rays, MeV electrons or photons) are in good agreement, whereas for higher doses larger variations are reported. The (60)Co gamma-ray data here for doses up to 1.5 Gy, using a low-yield serum batch and only one medium change, are in agreement with published frequencies of neoplastic transformation of human hybrid cells. For 3.4 MeV alpha-particles (LET = 124 keV/mum) and 0.565 MeV monoenergetic neutrons relative to low doses of (60)Co gamma-rays, a maximum relative biological effectiveness (RBE(M)) of 2.8 +/- 0.2 and 1.5 +/- 0.2, respectively, was calculated. Surprisingly, at higher doses of (60)Co gamma-rays lower frequencies of neoplastic transformation were observed. This non-monotonic dose relationship for neoplastic transformation by (60)Co gamma-rays is likely due to the lack of a G2/M arrest observed at low doses resulting in higher transformation frequencies per dose, whereas the lower frequencies per dose observed for higher doses are likely related to the induction of a G2/M arrest.

Suppression of NF-κB Activity by Parthenolide Induces X-Ray Sensitivity through Inhibition of Split-Dose Repair in TP53 Null Prostate Cancer Cells

We have shown that parthenolide, a sesquiterpene lactone, is a radiation sensitizer for human CGL1 hybrid cells that have constitutively activated NF-kappaB and wild-type p53. Since many malignant cells have nonfunctional p53, we investigated whether parthenolide could alter the X-ray sensitivity of PC-3 prostate cancer cells, a p53 null cell line with constitutively activated NF-kappaB. The addition of 5 microM parthenolide induced non-apoptotic cell death, inhibited PC-3 proliferation, and increased the population doubling time from 23+/-1 h to 49+/-4 h. Parthenolide also inhibited constitutive and radiation-induced NF-kappaB binding activity and enhanced the X-ray sensitivity of these p53 null PC-3 cells by a dose modification factor of 1.7. Cell cycle analysis of PC-3 cells treated with parthenolide showed only small alterations in G1 and G2/M cells, and these appeared to be insufficient to explain the observed radiosensitization. Split-dose studies using clinically relevant 2- and 4-Gy fractions demonstrated that parthenolide completely inhibited split-dose repair in PC-3 cells. We hypothesized that inhibition of NF-kappaB activity by parthenolide was responsible for the observed X-ray sensitization and inhibition of split-dose repair. To test this hypothesis, we knocked down the expression of NF-kappaB p65 protein, an active component of NF-kappaB in both PC-3 and CGL1 cells, by siRNA. Inhibition of NF-kappaB activity by knockdown of p65 increased radiation sensitivity and completely inhibited split-dose repair in both cell lines in a nearly identical manner as parthenolide treatment alone. Treating p65-depleted PC-3 cells with 5 microM parthenolide did not further increase their radiation sensitivity or the inhibition of split-dose repair. We propose that the suppression of radiation-induced NF-kappaB activity by parthenolide leads to X-ray sensitization through inhibition of split-dose repair in p53 null PC-3 prostate cancer cells.

Parthenolide Sensitizes Cells to X-Ray-Induced Cell Killing through Inhibition of NF-κB and Split-Dose Repair

Human cancers have multiple alterations in cell signaling pathways that promote resistance to cytotoxic therapy such as X rays. Parthenolide is a sesquiterpene lactone that has been shown to inhibit several pro-survival cell signaling pathways, induce apoptosis, and enhance chemotherapy-induced cell killing. We investigated whether parthenolide would enhance X-ray-induced cell killing in radiation resistant, NF-kappaB-activated CGL1 cells. Treatment with 5 microM parthenolide for 48 to 72 h inhibited constitutive NF-kappaB binding and cell growth, reduced plating efficiency, and induced apoptosis through stabilization of p53 (TP53), induction of the pro-apoptosis protein BAX, and phosphorylation of BID. Parthenolide also enhanced radiation-induced cell killing, increasing the X-ray sensitivity of CGL1 cells by a dose modification factor of 1.6. Flow cytometry revealed that parthenolide reduced the percentage of X-ray-resistant S-phase cells due to induction of p21 waf1/cip1 (CDKN1A) and the onset of G1/S and G2/M blocks, but depletion of radioresistant S-phase cells does not explain the observed X-ray sensitization. Further studies demonstrated that the enhancement of X-ray-induced cell killing by parthenolide is due to inhibition of split-dose repair.

Experimental techniques for studying bystander effects in vitro by high and low-LET ionising radiation

Ionising radiation can induce responses within non-exposed neighbouring (bystander) cells which potentially have important implications on the estimates of risk from low dose or low dose rate exposures of ionising radiations. A range of strategies have been developed for investigating bystander effects in vitro for both high-LET alpha particles or low-LET ultrasoft X rays using either partial shielding (grids, half-shields and slits) or by using a co-culture system where two physically separated populations of cells can be cultured together, allowing one population of cells to be irradiated while the second population remains unirradiated. The techniques described provide a useful tool to study bystander effects and complement microbeam studies. Studies using these systems show significant increases in the unirradiated bystander cells for various end points including the induction of chromosomal instability in haemopoetic stem cells and transformation in CGL1 cells.

A Radiation-Induced Acute Apoptosis Involving TP53 and BAX Precedes the Delayed Apoptosis and Neoplastic Transformation of CGL1 Human Hybrid Cells

Exposing CGL1 (HeLa x fibroblast) hybrid cells to 7 Gy of X rays results in the onset of a delayed apoptosis in the progeny of the cells 10 to 12 cell divisions postirradiation that correlates with the emergence of neoplastically transformed foci. The delayed apoptosis begins around day 8 postirradiation and lasts for 11 days. We now demonstrate that the delayed apoptosis is also characterized by the appearance of approximately 50-kb apoptotic DNA fragments and caspase 3 activation postirradiation. In addition, we confirm that stabilization of TP53 and transactivation of pro-apoptosis BAX also occurs during the delayed apoptosis and show that anti-apoptosis BCL-X(L) is down-regulated. To test whether the delayed apoptosis was due to a nonfunctional acute TP53 damage response in CGL1 cells, studies of acute apoptosis were completed. After irradiation, CGL1 cells underwent an acute wave of apoptosis that involves TP53 stabilization, transactivation of BAX gene expression, and a rapid caspase activation that ends by 96 h postirradiation. In addition, the acute onset of apoptosis correlates with transactivation of a standard wild-type TP53-responsive reporter (pG13-CAT) in CGL1 cells after radiation exposure. We propose that the onset of the delayed apoptosis is not the result of a nonfunctional acute TP53 damage response pathway but rather is a consequence of X-ray-induced genomic instability arising in the distant progeny of the irradiated cells.

The neoplastic transformation potential of mammography X rays and atomic bomb spectrum radiation.

Considerable controversy currently exists regarding the biological effectiveness of 29 kVp X rays which are used for mammography screening. This issue must be resolved to enable proper evaluation of radiation risks from breast screening. Here a definitive assessment of the biological effectiveness of 29 kVp X rays compared to the quality of radiation to which the atomic bomb survivors were exposed is presented for the first time. The standard radiation sources used were (a) an atomic bomb simulation spectrum and (b) 2.2 MeV electrons from a strontium-90/yttrium-90 (90Sr/90Y) radioactive source. The biological end point used was neoplastic transformation in vitro in CGL1 (HeLa x human fibroblast hybrid) cells. No significant difference was observed for the biological effectiveness of the two high-energy sources for neoplastic transformation. A limiting relative biological effectiveness (RBE(M)) of 4.42 +/- 2.02 was observed for neoplastic transformation by 29 kVp X rays compared to these two sources. This compares with values of 4.67 +/- 3.93 calculated from previously published data and 3.58 +/- 1.77 when the reference radiation was 200 and 220 kVp X rays. This suggests that the risks associated with mammography screening may be approximately five times higher than previously assumed and that the risk-benefit relationship of mammography exposures may need to be re-examined.

Homozygous deletions within the 11q13 cervical cancer tumor-suppressor locus in radiation-induced, neoplastically transformed human hybrid cells.

Studies on nontumorigenic and tumorigenic human cell hybrids derived from the fusion of HeLa (a cervical cancer cell line) with GM00077 (a normal skin fibroblast cell line) have demonstrated "functional" tumor-suppressor activity on chromosome 11. It has been shown that several of the neoplastically transformed radiation-induced hybrid cells called GIMs (gamma ray induced mutants), isolated from the nontumorigenic CGL1 cells, have lost one copy of the fibroblast chromosome 11. We hypothesized, therefore, that the remaining copy of the gene might be mutated in the cytogenetically intact copy of fibroblast chromosome 11. Because a cervical cancer tumor suppressor locus has been localized to chromosome band 11q13, we performed deletion-mapping analysis of eight different GIMs using a total of 32 different polymorphic and microsatellite markers on the long arm (q arm) of chromosome 11. Four irradiated, nontumorigenic hybrid cell lines, called CONs, were also analyzed. Allelic deletion was ascertained by the loss of a fibroblast allele in the hybrid cell lines. The analysis confirmed the loss of a fibroblast chromosome 11 in five of the GIMs. Further, homozygous deletion (complete loss) of chromosome band 11q13 band sequences, including that of D11S913, was observed in two of the GIMs. Detailed mapping with genomic sequences localized the homozygous deletion to a 5.7-kb interval between EST AW167735 and EST F05086. Southern blot hybridization using genomic DNA probes from the D11S913 locus confirmed the existence of homozygous deletion in the two GIM cell lines. Additionally, PCR analysis showed a reduction in signal intensity for a marker mapped 31 kb centromeric of D11S913 in four other GIMs. Finally, Northern blot hybridization with the genomic probes revealed the presence of a novel >15-kb transcript in six of the GIMs. These transcripts were not observed in the nontumorigenic hybrid cell lines. Because the chromosome 11q13 band deletions in the tumorigenic hybrid cell lines overlapped with the minimal deletion in cervical cancer, the data suggest that the same gene may be involved in the development of cervical cancer and in radiation-induced carcinogenesis. We propose that a gene localized in proximity to the homozygous deletion is the candidate tumor-suppressor gene.

Differential effectiveness of solar UVB subcomponents in causing cell death, oncogenic transformation and micronucleus induction in human hybrid cells

Purpose : (1) To determine the biological effectiveness of two solar ultraviolet (UVB) spectra with different lower wavelength thresholds for oncogenic transformation and micronucleus induction in CGL1 cells; (2) to investigate whether the action spectra for short- and long-term effects are similar; and (3) to investigate possible links between transformation and other delayed effects. Material and methods : Two spectra were derived from a solar UV simulator by using two filters: the first transmitted radiation with 5 > 284 nm, the second with 5 > 293 nm. The resulting spectra have the same UVA, but different UVB components (5 between 284 and 320 nm, 19 W m m 2, and 5 between 293 and 320 nm, 13 W m m 2) . CGL1 cells were irradiated with 466 J m m 2 with 5 > 284 nm and 1582 J m m 2 with 5 > 293 nm. These doses were approximately equilethal. The endpoints examined were oncogenic transformation, and centromere-positive and -negative micronucleus frequencies in the directly irradiated cells and in transtheir progeny. Results : At equilethal doses, the oncogenic transformation frequency in the directly irradiated cells was greater by a factor of at least 7 for 5 > 284 nm irradiation compared with 5 > 293 nm. The micronucleus induction frequency was also significantly higher with the 5 > 284 spectrum. Consistent with our previous findings, no delayed micronucleus formation was found in the progeny of cells exposed to 5 > 293 nm, while a threefold elevation above controls was seen in the progeny of cells exposed to 5 > 284 nm irradiation. This was also the case for formation of micronuclei with a centromere. Conclusions : It was found that: (1) for equilethal doses the 5 > 284 nm spectrum was more biologically effective than the 5 > 293 nm spectrum for induction of oncogenic transformation and micronucleus formation; and (2) the higher effectiveness of the 5 > 284 nm spectrum found at equilethal doses for delayed effects in the progeny of irradiated cells resembles that found for transformation. The results suggest that the UVB action spectrum for cell killing is different from that of some delayed effects, and from that of transformation.

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.

Solar UV radiation: differential effectiveness of UVB subcomponents in causing cell death, micronucleus induction and delayed expression of heritable damage in human hybrid cells

Purpose : To determine the effectiveness of two UV spectra with different UVB components for cell kill and micronucleus induction in irradiated human HeLa ×skin fibroblast (CGL1) hybrid cells and their progeny. To determine the presence of reactive oxygen species (ROS) in the progeny of the irradiated cells at various post-irradiation times and their relationship with induced delayed biological effects. Material and methods : A commercial solar ultraviolet simulator was used. Two different filters were employed: the first transmitted radiation with λ>284nm and the second radiation with λ>293nm. The resulting spectra have different UVB components (λbetween 284 and 320nm, 19 W/m 2, and between 293 and 320nm, 13 W/m 2) and the same UVA component (λbetween 320 and 400nm, 135 W/m 2) . CGL1 cells were irradiated with various doses. Clonogenic survival and micronucleus formation were scored in the irradiated cells and their progeny. ROS were detected by incubation of cultures at various post-irradiation times with dichlorodihydrofluorescein diacetate followed by flow cytometric measurement of the final product, dichlorofluorescein. Results : The biological effectiveness of the λ>284nm spectrum was higher by a factor of 3 compared to the λ>293nm spectrum for cell kill, and by a factor of 5 for micronucleus induction. No delayed cell death or micronucleus formation was found in the progeny of cells exposed to λ>293nm, while a large and dose-dependent effect was found in the progeny of cells exposed to λ>284nm for both of these endpoints. ROS levels above those in unirradiated controls were found only in the progeny of cells exposed to the λ>284nm spectrum. Conclusions : The spectrum with λ>284nm was more effective than that with λ>293nm for induction of cell kill and micronucleus formation in the directly irradiated cells as well as induction of delayed effects in the progeny in the form of delayed reproductive death and micronucleus formation. The presence of ROS in the progeny of the irradiated cells may be the cause of the delayed effects.

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.

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.

Enhanced expression of glucose transporter GLUT3 in tumorigenic HeLa cell hybrids associated with tumor suppressor dysfunction.

Previous studies on human cell hybrids between HeLa and normal human fibroblasts have indicated that the tumorigenicy may be controlled by a putative tumor suppressor gene on chromosome 11. We previously demonstrated a twofold increase in glucose uptake with a reduced Km by tumorigenic HeLa cell hybrids which expressed a highly glycosylated GLUT1. In this study, we reported that a tumorigenic cell hybrid, CGL4, also expressed a glucose transporter isoform, GLUT3, that was undetectable in nontumorigenic CGL1 cells. The expression of GLUT3 together with GLUT1 of 70 kDa was also evident in three gamma-ray-induced tumorigenic clones isolated from CGL1 cells, while control nontumorigenic irradiated cells expressed 50 kDa GLUT1 alone. In accordance with this, GLUT3 mRNA was specifically expressed in tumorigenic cell hybrids. To examine the role of GLUT3, clones which stably overexpress GLUT3 were developed from both CGL1 and CGL4 cells. In these transfectants, the affinity for 2-deoxyglucose markedly increased, in parallel with the amount of expressed GLUT3 irrespective of its N-glycosylation state. These results suggest that the enhanced GLUT3 expression in HeLa cell hybrids associated with the tumorigenic phenotypes may account for the increased affinity for 2-deoxyglucose. Possible roles of the putative tumor suppressor in control of gene expression and glucose uptake is discussed.

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.

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.

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 γ-ray-induced tumorigenic mutants (GIMs) isolated from CGL1 cells as expressing a tumor-associated surface antigen, intestinal alkaline phosphatase. In contrast, GLUT1 in the γ-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.