Molecular detection and genotyping of human papillomavirus in cervical carcinoma biopsies in an area of high incidence of cancer from Moroccan women.

Cervical cancer is a leading cause of cancer-related deaths in developing countries, and the human papillomavirus (HPV) is linked etiologically to cervical cancer. Eighty nine cervical carcinoma biopsies collected from women visiting the Oncologic Center in Casablanca (Centre Hospitalier Universitaire Ibn Rochd, Morocco) for cervical cancer symptoms, were screened for HPV DNA by polymerase chain reaction amplification with subsequent typing by hybridization with specific oligonucleotides for HPV types 16, 18, 31, 33, 45, and 59. Using very high stringency hybridization the HPV types could be easily distinguished. After preliminary clinical sorting, 92% (82/89) of the samples were found to be HPV-positive. Among the samples infected by a single HPV, type 16 was the most frequent 36.6% (30/82) of the positive samples, followed by HPV 18; 19.5% (16/82). Double or even multiple infections by the different HPV types were also detected (35.5% of the positive samples); dual infections were the more frequent, with the following combinations of HPVs: HPV16/HPV18 (21% of the positives samples) and HPV16/HPV45 (8.5%).

Role of glucagon in the control of heat production in ducklings.

Glucagon is known to be a central modulator of neural activity and a peripheral thermogenic effect. The purpose of this study was to better understand the role of glucagon in the control of heat production, shivering and particularly as a mediator of nonshivering thermogenesis (NST) in ducklings. In order to study the mechanism of NST, an intracerebroventricular (i.c.v.) injection of glucagon (10(-7) M) in to thermoneutral (TN), chronically glucagon treated (GT) and cold acclimatized (CA) ducklings exposed to acute cold (4 degrees C) or a thermoneutrality (25 degrees C), was performed. At 25 degrees C ambient temperature (Ta), the metabolic rate (MR) remained unchanged after glucagon injection. At 4 degrees C Ta i.c.v. glucagon injection, no significant change in MR was observed in GT and CA ducklings during 160 min of cold exposure, whereas there was 63% decrease in MR in (TN) ducklings (5.02 +/- 0.1 2 vs 7.91 +/- 0.1 4 W/kg(-1) p < 0.05). Shivering activity was completely suppressed in TN and GT ducklings after glucagon administration. The NST was estimated to be 3.26 W/kg. This findings suggest that glucagon administered into the brain has no thermogenic effect but could be involved in the central control of somatic motricity, and here we demonstrated for the first time, of our knowledge, that central glucagon have a role in the development of nonshivering thermogenesis during prolonged cold via an inhibition of shivering in birds.

Effects of interleukin-1 alpha on DNA repair in human ovarian carcinoma (NIH:OVCAR-3) cells: implications in the mechanism of sensitization of cis-diamminedichloroplatinum(II).

The cytokine interleukin-1 alpha (IL-1 alpha) showed a cytostatic effect on human ovarian carcinoma cells and significantly enhanced the antiproliferative activity of cis-diamminedichloroplatinum(II) (cisplatin) toward the NIH:OVCAR-3 tumor cell line in culture. The factor of sensitization was 15-20-fold. The maximum levels of sensitization were observed both with simultaneous exposure to cisplatin and IL-1 alpha and with 24-hr pretreatment with IL-1 alpha. Synergy between these agents was diminished when cells were pretreated with an IL-1 alpha-specific receptor antagonist, indicating that synergistic interaction was receptor mediated. Using atomic absorption spectroscopy, we evaluated the cellular accumulation of cisplatin and the DNA platination; the results showed that IL-1 alpha increased cellular accumulation of cisplatin and DNA platination. Cisplatin did not affect IL-1 alpha accumulation in NIH:OVCAR-3 cells. Further studies showed that IL-1 alpha reduced the removal of platinum from DNA. These results strongly suggest that IL-1 alpha inhibits DNA repair, and this decrease in DNA repair may explain, in part, the strong synergistic interaction between IL-1 alpha and cisplatin in NIH:OVCAR-3 cells.

Mechanisms of resistance to ansamycin antibiotics in human breast cancer cell lines.

We recently reported that multidrug-resistant, P-170 glycoprotein-positive, Adriamycin-selected, human breast tumor (MCF7/ADRR) cells were resistant to the benzoquinonoid ansamycin antibiotics geldanamycin (GL) and herbimycin A (HA) and that significantly fewer hydroxyl radicals were formed in resistant cells. We have carried out additional studies to define the mechanisms of cytotoxicity of and resistance to GL and HA, by directly examining the interactions of these drugs with P-170 glycoprotein using photoaffinity labeling. We found that both GL and HA inhibited binding of azidopine to P-170 glycoprotein in a dose-dependent manner. We have developed a 10-fold GL-resistant cell line (MCF7/GLR) by continuous drug exposure. Our studies indicated no significant differences in free radical formation between wild-type MCF7 cells and MCF7/GLR cells. Uptake and efflux studies indicated a small decrease in the GL accumulation but no difference in the efflux of GL in these cells. Verapamil had no effect on cellular accumulation of GL in wild-type MCF7 cells or MCF7/GLR cells. Verapamil significantly increased the accumulation of GL in MCF7/ADRR cells and enhanced GL cytotoxicity 12-fold, suggesting that GL interacted with the P-170 glycoprotein. Using reverse transcription-polymerase chain reaction, we found no expression of the mdr1 gene; however, expression of the multidrug resistance-associated protein was about 2-fold higher in MCF7/GLR cells. Taken together, these studies indicate that the mechanisms of GL resistance are multifactorial. Although decreased free radical formation may not play a significant role in low levels of GL resistance, e.g., in MCF7/GLR cells, both overexpression of mdr1 and decreased free radical formation contribute to GL resistance in highly resistant cells such as MCF7/ADRR cells.

Potentiation of antitumor activities of carboplatin and camptothecin by interleukin-1 alpha against human ovarian carcinoma in vivo.

Interleukin-1 alpha significantly potentiated the cytotoxicity of carboplatin (8-fold) and camptothecin (4-fold) during simultaneous drug exposure in human ovarian NIH: OVCAR-3 cancer cells in vitro. Treatment of human ovarian tumor cells grown as xenografts in nude mice with IL-1 alpha followed by either carboplatin or CTP-11 at minimally toxic doses significantly (2-3-fold and 7-fold for carboplatin and CTP-11, respectively) enhanced antitumor activity of either agent alone, indicating that IL-1-alpha-drug combinations may be potentially more effective for the treatment of ovarian tumors, including those difficult to cure in the clinic.

Inhibition of DNA repair and sensitization of cisplatin in human ovarian carcinoma cells by interleukin-1 alpha.

Interleukin-1 alpha induced an increase in both the cellular accumulation of cis-diamminedichloroplatinum (II) (cisplatin) and DNA platination and significantly reduced the removal of platinum from DNA of human ovarian (NIH: OVCAR-3) carcinoma cells in culture. The combinations of IL-1 alpha and cisplatin were highly synergistic against these ovarian carcinoma cells and maximum levels of sensitization (15-20-fold) were observed during simultaneous exposure of cisplatin and IL-1 alpha. IL-1 alpha specific receptor antagonist decreased this synergy. These results strongly indicate that IL-1 alpha inhibits DNA repair, and this inhibition of DNA repair may explain, in part, a strong synergistic interaction between IL-1 alpha and cisplatin in NIH: OVCAR-3 cells.

Doxorubicin-induced oxygen free radical formation in sensitive and doxorubicin-resistant variants of rat glioblastoma cell lines [corrected and republished erratum originally printed in FEBS Lett 1993 May 17;322(3):295-8].

We have studied the formation of hydroxyl radical (OH.) induced by doxorubicin in a series of doxorubicin- or vincristine-selected variants of C6 rat glioblastoma cells in culture by electron-spin resonance spectroscopy using 5,5'-dimethyl-1-pyrroline-1-oxide as a spin trap. Wild-type cells, sensitive to doxorubicin, exhibited in the presence of this drug a concentration-dependent OH. formation which could be inhibited by preincubation with superoxide dismutase, catalase or an antibody against cytochrome P450-reductase. In highly doxorubicin-resistant cells, OH. formation was reduced to about 20% of the level obtained in sensitive cells. In cells presenting a very low level of resistance to doxorubicin or in cells selected with vincristine, both presenting a pure multidrug-resistant phenotype, OH. formation was identical to that obtained in sensitive cells. In cells of intermediate resistance or in revertant cells, intermediate levels of OH. formation were obtained. Protection against OH. formation and action can be identified at the levels of superoxide dismutase and glutathione peroxidase activities, which are both enhanced in the resistant cells.

Doxorubicin-induced lipid peroxidation and glutathione peroxidase activity in tumor cell lines selected for resistance to doxorubicin.

Doxorubicin-induced lipid peroxidation was evaluated in four human or murine cell strains in culture and in their doxorubicin-resistant variants, by the quantification of malondialdehyde produced after a 2-h incubation of cells with the drug. Significantly increased malondialdehyde levels were obtained 24 h after doxorubicin treatment in three of the wild-type cell lines with doses as low as 0.05-0.1 micrograms/ml, which is within an order of magnitude of the concentration of the drug which inhibits cell growth by 50%. This production of malondialdehyde was abolished in two doxorubicin-resistant strains, even with high doses of drug (100-300 micrograms/ml), but was maintained in the third resistant line. No malondialdehyde production was observed in the fourth cell line, sensitive or resistant. It is remarkable that an enhancement of selenium-dependent and non-selenium-dependent glutathione peroxidase activities was exhibited during the acquisition of resistance to doxorubicin in the two first lines, but not in the third, whereas a constitutively high non-selenium-dependent glutathione peroxidase activity existed in the doxorubicin-sensitive and doxorubicin-resistant variants of the fourth cell line. Gene expression of selenium-dependent glutathione peroxidase and of glutathione S-transferase pi, which is known partially to bear a non-selenium-dependent glutathione peroxidase activity, were correlated with the corresponding enzyme activities. It appears, therefore, that the already known enhancement of glutathione peroxidase activity and expression in doxorubicin-resistant cell lines has a quantifiable consequence upon doxorubicin-induced lipid peroxidation and may have consequences in the mechanism of resistance to this drug.

Measurement of doxorubicin-induced lipid peroxidation under the conditions that determine cytotoxicity in cultured tumor cells.

We have investigated doxorubicin-induced lipid peroxidation by the measure of malondialdehyde (MDA) formation in rat glioblastoma cells and human breast carcinoma cells in culture. There was a significant production of MDA when the cells were incubated with pharmacologically relevant doxorubicin concentrations, i.e., concentrations that produce a significant cytotoxicity (0.1 micrograms/ml). At equitoxic doses, vincristine provided no lipid peroxidation, indicating that MDA formation is not a consequence of cell death. Doxorubicin-induced lipid peroxidation was maximal 24 h after incubation of the cells with doxorubicin, indicating that a delay was necessary for the free radical-mediated membrane damage induced by doxorubicin. In the presence of alpha-tocopherol in the culture medium, the doxorubicin-induced MDA formation was inhibited. The development of this method will help in defining the role of free radicals and lipid peroxidation in the cytotoxicity of doxorubicin.

Chromosomal modifications of a rat glioblastoma cell line during the acquisition and reversal of doxorubicin resistance.

We have studied the cytogenetic alterations occurring during the development and reversal of doxorubicin resistance in a clonal line of rat glioblastoma cells. We have observed during the acquisition of resistance an increase in the modal number of chromosomes, from 42 to 60, and the occurrence, in 90% of the mitoses, or large metacentric markers(s) which were infrequent in the sensitive line. This was associated with a net increase in total DNA amount per cell, from 5.3 to 8.3 pg. During reversal of resistance by 2 years culture without drug of the most resistant line, we observed a rapid decrease of the chromosome number as well as of the DNA content per cell; however, the large metacentric marker(s) were still present in 40% of the mitoses after 9 months of reversal, when the remaining resistance was only 4-fold. In situ hybridization of the chromosomes with a probe for the mdr gene revealed that the average number of stained chromosomes rose from 7% in the sensitive line to 38% in the most resistant line; however, only 9% of the silver grains were detected on the large metacentric markers. We conclude that important chromosome rearrangements occurred during the acquisition of resistance to doxorubicin, leading to a random distribution of the mdr gene in the genome.

Development of mechanisms of protection against oxidative stress in doxorubicin-resistant rat tumoral cells in culture.

We have compared some mechanisms involved in the defense against doxorubicin-induced free radical damage in rat hepatoma and glioblastoma cell lines and their doxorubicin-resistant variants presenting an overexpression of the multidrug resistance gene. Immediate in vivo production of malondialdehyde was minor and was not different in sensitive and resistant cells. Alpha-tocopherol was undetectable in all cell lines. Glutathione levels were not different in sensitive and resistant cells and these levels did not vary upon doxorubicin treatment. Resistant cells exhibited either a 50% decrease (hepatoma) or a 25% increase (glioblastoma) of glutathione-S-transferase activity. Glutathione reductase presented no important change upon acquisition of resistance. In contrast, selenium-dependent glutathione peroxidase activity was consistently 2-6-fold increased in the resistant cells, which suggests a magnification of protection mechanisms against hydroxyle radical formation from H2O2 in resistant cells. Depletion of glutathione levels by buthionine sulfoximine sensitized hepatoma resistant cells to doxorubicin, but had no effect on doxorubicin cytotoxicity to glioblastoma cells.

Alteration of ganglioside composition and metabolism in doxorubicin-resistant rat tumoral cells.

We have investigated the ganglioside levels, composition and metabolism in two lines of doxorubicin-resistant cells and in the corresponding wild strains, the C6 rat glioblastoma and the HTC rat hepatoma. The only ganglioside present was GM3, and its level was increased 2-fold in C6 resistant cells and decreased nearly 2-fold in HTC resistant cells. A decrease of cytidine 5'-monophospho-N-acetylneuraminic acid:galactosylglucosylceramide sialyltransferase activity was observed in both resistant lines as compared to sensitive ones, and could not, therefore, explain the increase in the GM3 level observed in the C6 resistant line. Alterations of acid neuraminidase activity were also observed; a 5-fold decrease was noticed in the C6 resistant line and could account for the increase in the GM3 level observed in these cells; in contrast, a 2-fold increase of acid neuraminidase activity was noticed in the HTC resistant cells: together, with reduced synthesis, it could explain the decrease in the GM3 level observed in these cells. No alterations of exogenous ganglioside transport was exhibited by the C6 resistant cells.