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Reversal of the temperature-shift-induced growth restriction of a temperature-sensitive simian virus 40 T-antigen-transformed human fibroblast cell line by treatment with retinoic acid.

ABSTRACT: We previously established a human fibroblast cell line, HFL 6-2, that contains a temperature sensitive simian virus 40 (SV40) T antigen, permitting cell growth at 35 degrees C but restricting growth at 39 degrees C. p21 (Waf1/Cip1) was significantly induced by temperature shifts in HFL 6-2 cells. Here we show that all-trans-retinoic acid (RA) treatment prevented the growth restriction of HFL 6-2 cells at 39 degrees C. In the presence of RA, HFL 6-2 cells proliferated into sizeable colonies even at 39 degrees C. [3H]Thymidine incorporation and flow cytometry analysis revealed that cells exposed to RA maintained DNA synthesis at 39 degrees C. Prevention of growth restriction by RA was correlated with a lack of induction of p21 at the transcription level. These observations suggest that RA may prevent the senescence process by repressing p21 gene expression, and perturb the growth regulation of somatic cells.

SUBMITTER: Tsao YP

PROVIDER: S-EPMC1217543 | biostudies-other | 1996 Aug

REPOSITORIES: biostudies-other

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Project description:Five immortalized brain capillary endothelial cell lines (TM-BBB1-5) were established from 3 transgenic mice harboring temperature-sensitive simian virus 40 large T-antigen gene (Tg mouse). These cell lines expressed active large T-antigen and grew well at 33 degrees C with a doubling time of about 20 to 30 hours. TM-BBBs also grew at 37 degrees C but not at 39 degrees C. However, growth was restored when the temperature of the culture was lowered to 33 degrees C. Although significant amounts of large T-antigen were shown to be present in the cell culture at 33 degrees C, there was less of this complex at 37 degrees C and 39 degrees C. TM-BBBs expressed the typical endothelial marker, von Willebrand factor, and exhibited acetylated low-density lipoprotein uptake activity. The alkaline phosphatase and gamma-glutamyltranspeptidase activity in TM-BBBs were -10% and 50% to 80% of brain capillary fraction of normal mice, respectively. D-mannitol transport in the both apical-to-basal and basal-to-apical directions across the TM-BBB was 2-fold greater than for inulin. TM-BBBs were found to express GLUT-1 but not GLUT-3, and exhibited concentration-dependent 3-O-methyl-D-glucose (3-OMG) uptake activity with a Michaelis-Menten constant of 6.59 +/- 1.16 mmol/l. Moreover, P-glycoprotein (P-gp) with a molecular weight of -170 kDa was expressed in all TM-BBBs. Both mdr1a and mdr1b mRNA were detected in TM-BBB4 using reverse transcription-polymerase chain reaction (RT-PCR) analysis. [3H]-Cyclosporin A uptake by TM-BBB was significantly increased in the presence of 100 micromol/l verapamil and vincristine, suggesting that TM-BBB exhibits efflux transport activity via P-gp. In conclusion, conditional brain capillary endothelial cell lines were established from Tg mice. This cell line expresses endothelial markers and transporters at the BBB and is able to regulate cell growth, due to the amount of active large T-antigen in the cell, by changing the culture temperature.

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Reversal of the temperature-shift-induced growth restriction of a temperature-sensitive simian virus 40 T-antigen-transformed human fibroblast cell line by treatment with retinoic acid.

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