Immunohistochemical analysis of proliferation and differentiation in organotypic cultures of cervical tumor cell lines

Researchers have previously demonstrated that organotypic cultures of cervical tumor cell lines exhibit morphological characteristics similar to the in vivo biopsies from which they were derived (Rader et al., 1990). Both the in vivo biopsy and organotypic culture appeared undifferentiated. We have extended these studies with immunohistochemical analysis using the proliferation and differentiation markers, proliferating cell nuclear antigen (PCNA) and involucrin, respectively, to evaluate in more detail the ability of cervical tumor cell lines to differentiate in organotypic culture. An HPV-immortalized keratinocyte cell line, PE-4, expressed PCNA in the lower half and involucrin in the upper half of the organotypic culture which is consistent with the characteristics of a preneoplastic lesion in vivo. The CC-1 cell line, derived from an invasive squamous cell carcinoma, appeared undifferentiated, but expressed involucrin in the upper half of the organotypic culture. This is the first observation of expression of a differentiation marker in an organotypic culture of a cervical tumor cell line. The other cervical tumor cell lines, SiHa and HeLa, derived from a squamous cell carcinoma, and an adenocarcinoma of the cervix, respectively, did not express detectable levels of involucrin or mucin. All three cervical tumor cell lines, CC-1, SiHa and HeLa, expressed PCNA throughout their entire thickness. The majority of nuclei in SiHa and HeLa cultures were PCNA-positive, while the CC-1 cell line exhibited a lower growth fraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of radiation-induced G2 delay potentiates cell death by apoptosis and/or the induction of giant cells in colorectal tumor cells with disrupted p53 function

We have previously identified a p53-independent apoptotic response that is delayed until 48-72 h after irradiation of colorectal adenoma and carcinoma cells. Because the delay appears to be in part due to a transient G2 cell cycle arrest, the importance of this checkpoint in the mechanism of ionizing radiation (IR)-induced death of colorectal tumor cells was investigated. An adenoma cell line with (282Arg-->Trp) mutant p53 (S/RG/C2) and a carcinoma cell line (PC/JW/FI) lacking p53 protein treated with 5 Gy IR in the presence of 1.5 mm caffeine (CAF) reduced IR-induced G2 arrest and increased the level of apoptosis (1.5-1.6-fold) 24 h after treatment. Increased IR apoptotic cell death with CAF significantly reduced IR cell survival over a 7-day period in S/RG/C2 and PC/JW/FI. To investigate whether CAF radiosensitization correlated with lack of wild-type (wt) p53, we studied transfected derivatives of an adenoma-derived cell line (PC/AA/C1), in which the endogenous wt p53 activity was disrupted by the expression of a dominant negative (273Arg-->His) p53 mutant protein (designated AA/273p53/B). This p53-defective cell line was also radiosensitized by CAF, whereas the vector control (AA/PCMV/D), which retained wt p53 activity, was not. In addition, as with the S/RG/C2 and PC/JW/FI cell lines, the 7-day IR cell survival was reduced significantly in AA/273p53/B compared with the vector control cell line. This suggests that radiosensitization by CAF and increased cell death is dependent on loss of wt p53 function. Interestingly, radiosensitization of the AA/273p53/B cell line was not associated with accelerated apoptosis but correlated with increased polyploid giant cells, which have been associated with disruption of cell cycle checkpoints and genomic instability. These results demonstrate that G2 checkpoint inhibition with CAF leads to preferential IR cell killing in cell lines in which wt p53 is inactivated and that this increased cell killing is not necessarily dependent on increased IR-induced apoptosis.     

Sensitivity of human prostatic carcinoma cell lines to low dose rate radiation exposure

PURPOSE: Low dose rate radioemitters, such as 125I, 103Pd, and 89Sr, have been used both for local and systemic treatment of prostate cancer. Most normal cells exposed to ionizing radiation characteristically activate cell cycle checkpoints, resulting in cell cycle arrest at the G1/S and G2/M transition points. Cancer cells are typically quite sensitive to radiation killing late in the G2 phase of the replicative cell cycle. Furthermore, most cancer cells accumulating at the G2/M transition point as a result of low dose rate radiation exposure appear to become sensitive to further low dose rate irradiation. For this reason, protracted exposure of cancer cells to low dose rate radiation has been proposed to result in increased cancer cell killing as compared with brief exposures of cancer cells to high dose rate radiation. Since many human prostatic carcinomas contain somatic genome alterations targeting genes which affect the cell cycle and radiation-associated cell cycle checkpoints, we evaluated the effects of low dose rate radiation exposure on the cell cycle and on clonogenic survival for various human prostatic carcinoma cell lines. MATERIALS AND METHODS: Human prostatic carcinoma cells from the LNCaP, DU 145, PC-3, PPC-1, and TSU-Pr1 cell lines were exposed to low dose rate (0.25 Gy/hour) or high dose rate (60 Gy/hour) radiation in vitro and then assessed for radiation cytotoxicity by clonogenic survival assay. Cell cycle perturbations following protracted exposure to low dose rate radiation were evaluated using flow cytometry. RESULTS: For LNCaP cells, low dose rate radiation exposure resulted in an accumulation of cells at both the G1/S and the G2/M cell cycle transition points. For DU 145, PC-3, PPC-1, and TSU-Pr1 cells, treatment with low dose rate radiation triggered G2/M cell cycle arrest, but not G1/S arrest. Unexpectedly, the cell cycle redistribution pattern phenotypes observed, G1/S and G2/M cell cycle arrest versus G2/M arrest alone, appeared to have little effect on low dose rate radiation survival. Furthermore, while PC-3, PPC-1, and TSU-Pr1 cells exhibited increased cytotoxic sensitivity to low dose rate versus fractionated high dose rate radiation treatment, DU 145 and LNCaP cells did not. CONCLUSIONS: Radiation-associated pertubations in replicative cell cycle progression were not dominant determinants of low dose rate radiation killing efficacy in human prostate cancer cell lines in vitro.     

Factors associated with the therapeutic efficacy of retinoic acids on malignant lymphomas

We recently reported the successful use of retinoic acids in the treatment of refractory lymphoma. The biologic determinants predicting response of lymphomas to retinoic acid remain unknown. This study was conducted to explore this question using in vitro models. Sensitivity of representative lymphoma cells to 13-cis-retinoic acid was determined. Sensitive and resistant cell lines were then compared for their baseline and/or retinoic-acid-regulated expression of total cellular retinoic acid binding protein, retinoic acid receptor (RAR)-alpha, RAR-beta, RAR-gamma mRNA, retinoid X receptor (RXR)-alpha, RXR-beta, RXR-gamma mRNA, transforming growth factor (TGF)-beta 1 and TGF-beta 1 receptors, and Fas (Apo-I) mRNA. The results showed that four of five T, two of three Hodgkin's, and none of six B cell lymphoma cell lines were sensitive (IC30 < 1.5 mmol/L) to 13-cis-retinoic acid. Further analyses revealed several of the above-mentioned parameters may be relevant to retinoic acid sensitivity. Baseline expression of TGF-beta 1 receptors was present in all of the five sensitive cell lines examined, but in only one of the four resistant cell lines. The correlation of Fas expression and retinoic acid sensitivity was good for B cell lines, but not apparent for T cell or Hodgkin's cell lines. On exposure to retinoic acid, an immediate and prolonged upregulation of RAR-alpha mRNA expression, lasting for more than 12 hours, occurred in all sensitive cell lines, but only minimal or transient induction was seen in resistant cells. Together, these data suggested that; 1) retinoic acid has a preferential effect on T cell and Hodgkin's lymphoma cell lines; 2) autoregulation of RAR-alpha by retinoic acids, and the presence of TGF-beta 1 receptors may be relevant to the response of lymphomas to treatment with retinoic acids.     

Radiation induced G1-block and p53 status in six human cell lines

Considerable attention has recently been focused on the fact that the tumor suppressor protein p53 is involved in the cellular response to radiation. In its wild-type form the protein appears to control a cell cycle checkpoint, preventing entry into S-phase following DNA damage. A number of authors observed a radiation induced G1-block in cells expressing wild-type p53, but not in p53 mutant cells. We obtained similar results with four human tumour cell lines as well as two strains of human fibroblasts, whose p53 status was ascertained at the protein as well as DNA levels. In addition to cell cycle delays in exponentially growing cell cultures, we have studied the possible role of the p53 in the transition from quiescence to active proliferation. Cells were irradiated after 6 days of serum-starvation and labelled with BrdU at different times after addition of fresh medium. Entry into S-phase was found to be delayed by several hours in the p53 wild-type cells, but no such effect was observed in the p53 mutants. Where a delay occurred, it was roughly proportional to the X-ray dose. Although it remains to be clarified, whether the cells were delayed only in G1 or also in G0, it is interesting to note that entry into S-phase can be delayed by irradiation in a quiescent state immediately before serum-stimulation, provided the cells are wild-type with respect to p53. Certain differences in the cell cycle response of transformed and untransformed cells were noted.     

Effects of irradiation on the expression of major histocompatibility complex class I antigen and adhesion costimulation molecules ICAM-1 in human cervical cancer

PURPOSE: We initiated studies to analyze the effects of high doses of gamma irradiation on the surface antigen expression of MHC Class I, Class II, and ICAM-1 on human cervical carcinoma cell lines. METHODS AND MATERIALS: The expression of surface antigens (MHC Class I, Class II, and ICAM-1) was evaluated by FACS analysis on two cervical cell lines at different time points, following their exposure to high doses of gamma irradiation (i.e., 25.00, 50.00, and 100.00 Gy). RESULTS: The CaSki and SiHa cervical cancer cells we analyzed in this study expressed variable levels of MHC Class I and ICAM-1 antigens, while Class II surface antigens were not detectable. Whereas irradiation doses of 25.00 Gy were not sufficient to totally block cell replication in both cell lines, exposure to 50.00 or 100.00 Gy was able to completely inhibit cell replication. Range doses from 25.00 to 100.00 Gy significantly and consistently increased the expression of all surface antigens present on the cells prior to irradiation but were unable to induce neoexpression of antigens previously not expressed by these cells (i.e., MHC Class II). Importantly, such upregulation was shown to be dose dependent, with higher radiation doses associated with increased antigen expression. Moreover, when the kinetic of this upregulation was studied after 2 and 6 days after irradiation, it was shown to be persistent and lasted until all the cells died. CONCLUSIONS: These findings may partially explain the increased immunogenicity of tumor cells following irradiation and may suggest enhanced immune recognition in tumor tissue in patients receiving radiation therapy.     

Radiation-enhanced expression of E6/E7 transforming oncogenes of human papillomavirus-16 in human cervical carcinoma

BACKGROUND: Human papillomavirus (HPV) infection represents the most important risk factor for cervical carcinoma. Levels of expression of E6 and E7 transforming oncoproteins of high risk HPV genotypes (i.e., HPV-16 and HPV-18) have been linked specifically to the mitotic activity of cervical carcinoma and appear to be necessary for maintaining the malignant phenotype. However, E6/E7 viral proteins recently have been reported to be effective tumor rejection antigens in animal models and humans. Radiation treatment represents a standardized and effective modality for contemporary cervical carcinoma therapy. However, although the physiologic and cellular changes associated with high doses of irradiation have been well documented it has been shown only recently that an increased synthesis of specific cellular proteins is observed after irradiation. In this study, the authors analyzed the effects of high doses of gamma irradiation on the expression of E6/E7 oncoproteins in HPV-16-infected cervical carcinoma cell lines. In addition, the effects of radiation on major histocompatibility complex (MHC) restriction elements also were studied. METHODS: The effect of high doses of gamma irradiation (i.e., 1250, 2500, 5000, and 10,000 centigray [cGy]) on the kinetics of E6/E7 oncoprotein expression in two HPV-16 positive cervical carcinoma cell lines (i.e., CaSki and SiHa) was evaluated by Northern blot analysis. In addition, the effect of radiation on the expression of MHC molecules also was studied by Northern blot and fluorescence activator cell sorter (FACS) analysis. RESULTS: Dose ranging from 1250 (sublethal) to 10,000 (lethal) cGy significantly increased the expression of E6/E7 oncoproteins as well as MHC Class I molecules in CaSki and SiHa cell lines when compared with untreated tumor cells. Both cell lines showed increased mRNA expression for MHC Class I molecules in a dose-dependent manner. E6/E7 oncoproteins also were up-regulated in a dose-dependent manner in the CaSki cell line, whereas in the SiHa cell line their expression plateau at 5000 cGy. When the kinetics of radiation-induced up-regulation of E6/E7 were studied, persistent up-regulation of the viral oncoproteins was noted for all doses of irradiation, with the lower and sublethal doses (i.e., 1250-2500 cGy) inducing the most significant enhancement. CONCLUSIONS: High doses of irradiation can induce a significant and long-lasting up-regulation of E6/E7 oncogenes and MHC Class I restriction elements on HPV positive cervical carcinoma cell lines. These effects by themselves suggest that irradiation could enhance local tumor immunogenicity in patients receiving radiation therapy. However, in contrast to this possible beneficial effect, sublethal tumor irradiation (up-regulating E6/E7 transforming oncoproteins) also could confer a significant growth advantage to radiation-resistant tumor cells. These findings, combined with the previously reported acquisition of a radiation-induced drug resistance, could provide a biologic basis for the poor prognosis of patients with cervical carcinoma recurrence after radiation therapy.     

Biologically active heteroarotinoids exhibiting anticancer activity and decreased toxicity

A series of retinoids, containing heteroatoms in a cyclic ring and called heteroarotinoids, were synthesized, and their biological activity was evaluated using tissue culture lines that have measurable responses to trans-retinoic acid (t-RA). Transglutaminase (TGase) was assessed in the human erythroleukemia cell line (GMO6141A) as an indicator of differentiation and apoptosis. Proliferation was evaluated in a human cervical cell line, CC-1, which exhibits dose-dependent alterations in growth rate in response to treatment with trans-retinoic acid. Activation of nuclear retinoic acid receptors was determined in a reporter cell line established from CC-1. The reporter line, called CC-B, contains a reporter gene controlled by a retinoic acid responsive element (RARE) and a thymidine kinase (tk) promoter. Treatment of the CC-B line with the heteroarotinoids resulted in a dose-responsive and retinoid-dependent regulation of reporter gene expression. The heteroarotinoids exhibited activity in all assays and correlated in a statistically significant manner between assays. RARE transactivation activity in CC-B cells correlated with induction of TGase in GMO6141A (R = 0.96) and with a decrease in the growth rate of CC-1 cells (R = -0.90). The ability of the selected heteroarotinoids to induce differentiation, inhibit proliferation, and activate nuclear receptors demonstrates the chemotherapeutic potential of these agents. In view of the biological activity cited, an in vivo toxicity study was conducted on male B6D2F1 mice with three heteroarotinoids, namely 8 [(2E,4E,6E)-3,7-dimethyl-7-(1,2,3,4-tetrahydro-4,4-dimeth ylthiochroman-6-yl)-2,4,6-heptatrienoic acid], 10 [(2E,4E,6E)-3,7-dimethyl-7-(1,2,3,4-tetrahydro-4,4-dimeth ylchroman-6-yl)-2, 4,6-heptatrienoic acid], and 13 [(E)-p-[2-(4,4-dimethylchroman-6-yl)propenyl]benzoic acid]. The mice were used with gavage of heteroarotinoids in corn oil [0.1, 0.2, 0.4, or 0.8 mg/kg] and with 0.01 or 0.05 mg/kg of TTNPB (5) [(E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1- propenyl]benzoic acid] as reference controls. The target organs affected in the mice by the three heteroarotinoids were those typically associated with t-RA (1) toxicity. The maximum tolerated dose (MTD) of 13 was 9.4 mg/kg/day, which was equal in toxicity to that of t-RA (1) and 1000-fold less toxic than TTNPB (5). The MTDs of 8 and 10 were 34 and 32 mg/kg/day, respectively, which is 3-fold less toxic than t-RA (1) and 3000-fold less toxic than TTNPB (5). The 3000-fold reduced toxicity, compared with only a 27% reduction biological activity of 8 and 10 with respect to that of TTNPB, observed in our assays indicates a good therapeutic ratio of these heteroarotinoids over the parent compound. The biological activity and reduced toxicity of these heteroartinoids demonstrate the potential efficacy as anticancer agents.     

Endothelium-dependent blunted membrane potential responses to ATP-sensitive K+ channel modulators in aortae from rats with cirrhosis

Gemcitabine, 2'2'-difluoro-2'-deoxycytidine, is an inhibitor of DNA synthesis and has been shown previously in vitro and in vivo to enhance the cytotoxic activity of radiation as well as some chemotherapeutic agents. Because gemcitabine has shown clinical activity on its own in several solid tumors traditionally treated with radiotherapy, it was of interest to optimize the combination of gemcitabine and radiation. To determine the optimal gemcitabine dose to combine with irradiation and to determine the effect of gemcitabine on tumor growth, mice bearing SA-NH tumors were treated with 2.5 to 600 mg/kg gemcitabine, and subsequent tumor growth was determined. At low doses, gemcitabine induced transient growth delay, whereas higher doses showed both cytotoxic and cytostatic activity. Flow cytometric, histological, and mitotic analyses of irradiated tumors showed that gemcitabine induced a dose-dependent inhibition of DNA synthesis and induction of apoptosis of cells in S phase. DNA synthesis recovered in cells at the G1-S boundary of the cell cycle in a dose-dependent manner, and a parasynchronous movement of cells through the cell cycle ensued. To determine the optimal schedule for gemcitabine administration in relation to irradiation, tumor-bearing mice were given a single 50 mg/kg dose of gemcitabine at various times before or after irradiation. Gemcitabine enhanced radioresponse in a time-dependent fashion. The highest enhancement factors for tumor growth delay (1.68-2.03) were observed when gemcitabine was administered 24-60 h before irradiation. Although gemcitabine reduced the radiation tumor control dose at all administration times used, the greatest enhancement of tumor radiocurability occurred when gemcitabine was administered 24 h before irradiation (dose modification factor of 1.54). Moreover, gemcitabine decreased the lung metastatic rate in mice with local tumor control from 73% in mice receiving radiation alone to 40% in mice receiving the combination (all combination times included). These results suggest that gemcitabine has strong radioenhancing properties and that the greatest interaction occurs when gemcitabine administration precedes irradiation by 24-72 h. Preliminary studies indicate that normal tissues recover more quickly than tumor tissues from gemcitabine treatment; thus, optimized scheduling of gemcitabine and irradiation may serve to improve the therapeutic ratio of the combination.     

IFNgamma inhibition of cell growth in glioblastomas correlates with increased levels of the cyclin dependent kinase inhibitor p21WAF1/CIP1

Glioblastoma is a highly aggressive form of brain cancer characterized by uncontrolled cell growth resulting from a loss of cell cycle regulation. In this study we determined the antiproliferative effects of interferon gamma (IFNgamma) on the glioblastoma cell lines T98G, SNB-19 and U-373, focusing on the ability of IFNgamma to increase levels of p21WAF1/CIP1, an important negative regulator of cell cycle events. IFNgamma was found to inhibit the growth of all cell lines, with inhibition ranging from 82.2% to 45.4%. Flow cytometry analysis showed that IFNgamma treatment caused a cell cycle delay in the G1 or S phases. The strength of this delay varied, correlating with the degree by which IFNgamma inhibited proliferation of each cell line. IFNgamma treatment increased the production of the cyclin dependent kinase inhibitor (CKI) p21WAF1/ CIP1 in all cell lines, the level and kinetics of production of which correlated with the degree and stage of inhibition of cellular proliferation. Further, immunoprecipitation of p21WAF1/CIP1 in complexes of p21WAF1/CIP1/cyclin-dependent kinase 2 (cdk2)/cyclin showed that the amount of p21WAF1/CIP1 in the complexes and the inhibition of cdk2-cyclin kinase activity correlated with the level of p21WAF1/CIP1 produced in the cells by IFNgamma. These results show that IFNgamma has significant antiproliferative effects on the glioblastoma cell lines and suggest that p21WAF1/CIP1 plays a role in mediating these effects.     

1,25-dihydroxyvitamin D3 and 9-cis-retinoic acid act synergistically to inhibit the growth of LNCaP prostate cells and cause accumulation of cells in G1

Recent studies have suggested that the active metabolite of vitamin D3, 1,25-dihydroxyvitamin D3, can inhibit the growth and/or induce the differentiation of a variety of cell types and that these characteristics might be useful in the treatment of some cancers. Retinoids also promote the differentiation and inhibit the growth of some cells. That the vitamin D receptor acts as a heterodimer with the retinoid X receptor (RXR) suggests that there may be functional interactions between 1,25-dihydroxyvitamin D3 and retinoids. In this study, we show that the combination of 1,25-dihydroxyvitamin D3 and 9-cis retinoic acid synergistically inhibits the growth of LNCaP prostate cancer cells. That this effect is mediated by RXR rather than retinoic acid receptors was shown using RXR- and retinoic acid receptor-specific ligands. The vitamin D3 analog, EB1089, inhibited growth more effectively than 1,25-dihydroxyvitamin D3 and also acted synergistically with 9-cis-retinoic acid. These treatments caused cells to accumulate in the G1 phase of the cell cycle, suggesting that 1,25-dihydroxyvitamin D3 can regulate one or more factors critical for the G1/S transition.     

A case cohort study of suicide in relation to exposure to electric and magnetic fields among electrical utility workers

BACKGROUND: All-trans retinoic acid is currently used in clinical trials in combination with tamoxifen to treat breast cancer, and 13-cis retinoic acid is used with a-interferon to treat metastatic endometrial cancer. We examined the effects of all-trans retinoic acid and 13-cis RA alone on endometrial adenocarcinoma (RL95-2) cells to investigate the cell biological mechanisms by which retinoic acid may reduce the metastatic phenotype and induce differentiation. METHODS: RL95-2 cells were seeded onto 4-chamber plastic slides and treated with 13-cis retinoic acid or all-trans retinoic at 0.5 microM, 1 microM and 5 microM doses for 90 minutes at 37 degrees C and stained for F-actin. RESULTS: Untreated RL95-2 cells exhibited staining of disrupted aggregates of F-actin only near the cell periphery. Cells treated with the three doses of 13-cis retinoic acid exhibited a dramatic reorganization of F-actin throughout the cells. When cells were treated with 0.5 microM all-trans retinoic acid, actin filaments reorganized. Cells treated with 1 microM all-trans retinoic acid and 5 microM all-trans retinoic acid displayed increased organization of F-actin and cell size increased. The percentage of S-phase cells increased at the high doses of retinoic acid treatment. This effect was apparently transient, since retinoic acid did not significantly affect cell growth. CONCLUSION: An organized cytoskeleton and an increase in cell size are associated with differentiation. We suggest that retinoic acid exerts its effects on these transformed cells by reorganizing actin filaments, and inducing differentiation, thus inducing a more stationary phenotype.     

Influence of ionizing radiation on proliferation, c-myc expression and the induction of apoptotic cell death in two breast tumour cell lines differing in p53 status

PURPOSE: To determine the capacity of ionizing radiation to inhibit proliferation, to suppress c-myc expression and to induce apoptotic cell death in the p53 wild-type MCF-7 cell line and the p53 mutated MDA-MB231 cell line. MATERIALS AND METHODS: Growth inhibition and cell killing were determined by cell number and trypan blue exclusion. Apoptosis was assessed through cell morphology and fluorescent end-labelling. c-myc expression was monitored by Northern blotting. RESULTS: Inhibition of cell proliferation by ionizing radiation was similar in both cell lines. MDA-MB231 cells accumulated in G2 while MCF-7 cells accumulated in both the G1 and G2 phases of the cell cycle after irradiation. There was no evidence of apoptosis in either cell line. In MCF-7 cells, growth inhibition correlated closely with an early dose-dependent suppression of c-myc expression; in MDA-MB231 cells, there was no correspondence between growth inhibition and a transient, dose-independent reduction in c-myc message. CONCLUSIONS: These findings suggest that in the absence of classical apoptotic cell death, radiosensitivity is not predictably related to the p53 status of the cell. While both p53 and c-myc may be linked to the DNA damage response pathway, neither p53 nor c-myc are essential for growth arrest in response to ionizing radiation.     

Is the Beck Depression Inventory reliable over time? An evaluation of multiple test-retest reliability in a nonclinical college student sample

The staurosporine-induced G1 cell cycle arrest was analyzed in a variety of cell lines which includes human tumor cell lines and oncogene-transformed NIH3T3 cell lines. All the cell lines which were sensitive to staurosporine-induced G1 arrest contained a functional retinoblastoma protein (pRB). However, when pRB-lacking fibroblast cells derived from pRB knockout mice were tested they were also sensitive to G1 arrest by staurosporine, indicating that the inactivation of pRB alone is not sufficient for the abrogation of staurosporine-induced G1 arrest. In searching for a common event caused by staurosporine, the cyclin-dependent kinase (CDK) inhibitor protein p27kip1 but not p21cip1 was found to accumulate after staurosporine treatment in all the cell lines examined. This accumulation occurred regardless of the induction of the G1 arrest. The result indicates that the accumulation of p27kip1 is the cell's primary response to staurosporine and that the capability of staurosporine to induce G1 arrest depends on the integrity of cell cycle regulatory components which are downstream of p27kip1.     

Absence of p53-dependent cell cycle regulation in pluripotent mouse cell lines

We examined the expression of p53 in three lines of pluripotent embryonal carcinoma (EC) and ES cells. p53 mRNA and protein levels were constitutively high in two lines but absent from one. In the P19 line of EC cells neither p53 protein nor mRNA was detected. The first intron of the p53 gene in these cells had been invaded by a murine leukemia virus and there was extensive hypermethylation of the p53 gene accompanying its inactivation. In all three cell lines, irradiation resulted in arrest of the cells in the G2 but not in the G1 phase of the cell cycle despite the induction of p21cip1 in the cell lines expressing p53. Thus, the chromosomal stability of EC and ES cells appears to be not dependent on the p53 protein and we interpret our results to suggest that these cells may require the deletion of p53 dependent cell cycle regulation in order to become immortalized.     

Effect of beta-carotene on cell cycle progression of human fibroblasts

The uptake of beta-carotene (BC) and its effect on the cell cycle progression of normal human fibroblasts in primary culture were investigated by using two different delivery methods: exposure to BC solubilized in the organic solvent tetrahydrofuran (THF) or to BC incorporated into dipalmitoylphosphatidylcholine (DPPC) liposomes. Cell cycle progression was evaluated by immunofluorescence detection and flow cytometric analysis of the proliferating cell nuclear antigen (PCNA). In contrast to THF, which induced a marked reduction in the number of cells in S phase and in the extent of PCNA immunolabeling, DPPC liposomes proved to be an effective delivery system that does not interfere with cell proliferation. Cellular uptake of 0.23 nmol/10(6) cells was found after 24 h incubation in BC-containing DPPC liposomes. This value increased to 1.2 nmol/10(6) cells after 72 h. After the first day of incubation, the number of cells in S phase was reduced by approximately 50%, with a consequent accumulation of cells in G1 phase. This effect was maintained up to 3 days incubation, with no detectable effects on cell viability. This cell cycle delay was found to be reversible, returning the percentage of cells in S phase to the control value 24 h after removal of BC from the medium. In order to determine whether the activity of BC could be attributed to the molecule itself or to its conversion into retinoids, the production of BC metabolites was assessed. Analysis of cellular levels of retinoids failed to demonstrate the presence of retinal, retinol, retinoic acid or retinyl esters during an incubation period of 6 days. These results suggest that in normal human fibroblasts, BC induces a cell cycle delay in the G1 phase and that this effect is independent of conversion to known retinoids.     

Concomitant treatment with mild hyperthermia, cisplatin and low dose-rate irradiation in human ovarian cancer cells sensitive and resistant to cisplatin

Hyperthermia has been shown to be an effective radiation sensitizer. Cisplatin has also been shown to cause radiosensitization. In part, the sensitization is through the inhibition of repair of radiation damage. In this study we have set out to combine low dose-rate irradiation (during which extensive repair occurs) with both cisplatin and hyperthermia to maximize the radiation sensitizing effect. Two human ovarian carcinoma cell lines, one parental (A2780S) and the other a cisplatin resistant derivative (A2780CP) cell line were used in these experiments. Long duration hyperthermia at 40 degrees C was combined with low concentrations of cisplatin (0.5-3 microg/ml) and low dose-rate irradiation (LDRI). The responses to the individual treatments showed that there was cross resistance in the two cell lines for cisplatin and radiation, but for hyperthermia the opposite effect was found. When all treatments were given concurrently the response was greater than the calculated response of all three individual treatments, indicating a synergistic interaction. The effect was greater in the cisplatin resistant cell line. The combination of mild hyperthermia, low dose cisplatin and LDRI are a good combination for potential clinical application. In addition, this could be a good approach to deal with cisplatin resistance.     

Chemistry of loteprednol etabonate and related steroids. II. Reactions at ring C and NMR structural studies of the resulting compounds

The effects of three inducers of differentiation, phorbol myristate acetate (PMA), retinoic acid (RA) and interferon-gamma (IFN-gamma), on the temporal regulation of vitamin D receptor (VDR) expression in HL-60 cells were analyzed by Northern blotting and immunofluorescence assays. VDR, at the protein level, expressed by 81% of uninduced cells, was reduced to 57% after 48 h of PMA or 96 h of RA treatment, preceded by growth inhibition and cell differentiation, evaluated by CD11b expression. Sorted CD11b positive cells in G0/G1 phase exhibited 53% the VDR content of CD11b negative cells (distributed throughout the cell cycle). PMA also induced an increase in PKC beta and PKC alpha mRNA and protein. Simultaneous exposure to PMA and sphingosine blocked stimulation of CD11b and PKC expression without affecting growth arrest and VDR down regulation. Similar effects were observed during sphingosine treatment. In IFN-gamma differentiated cells, the proportion of cells in G0/G1 phase was unchanged and VDR protein was unaltered as compared to uninduced cells. Control cells in G0/G1 expressed less VDR than cells in S and G2/M phases (74% and 59% respectively). All results suggest that in HL-60 cells, reduction of VDR expression is related to growth inhibition rather than to the differentiation process.     

Effect of the insulin-like growth factor I receptor on ionizing radiation-induced cell death in mouse embryo fibroblasts

We have investigated the effect of the insulin-like growth factor I receptor (IGF-IR) on ionizing radiation (IR)-induced cell death using the following two mouse embryo fibroblast cell lines: (i) R- cells with a null mutation of the IGF-IR gene, therefore expressing no endogenous IGF-IR; (ii) R+ cells derived from R- cells, a stable transfectant overexpressing the human IGF-IR. Numbers of R- cells began to detach from dishes and float into the medium about 48 h after 10 Gy of X-irradiation. Internucleosomal DNA fragmentation detected by agarose gel electrophoresis, which is characteristic of apoptosis, was observed in the floating R- cells, but not in the attached cells. Unexpectedly, morphological analysis of the floating cells 72 h after irradiation revealed that only about half of them showed apoptotic death and the rest showed a nonapoptotic, presumably necrotic, one. On the other hand, R+ cells retained more than 90% viability even 4 days after irradiation, and very few floating cells were observed. The G2 arrest was induced in both cell lines following irradiation and G2/M fractions similarly returned to normal levels by around 20 h after irradiation, indicating that the cell death which appeared thereafter in R- cells is mediated through mitosis. Significant induction of p53 following irradiation was not detected by Western blot analysis in either R- or R+ cells. Collectively, these results demonstrate that signal transduction pathways originating from the IGF-IR may be involved in preventing IR-induced apoptosis and necrosis without affecting cell cycle arrest or p53 pathways.