Heat treatment induces dephosphorylation of pRb and dissociation of T-antigen/pRb complex during transforming infection with SV40.

The effects of heat treatment on key cell cycle regulatory molecules is currently unknown. Using primary mouse kidney cell cultures induced to re-enter S-phase by transforming infection with SV40, the effects of thermal treatment on the phosphorylation state of the Retinoblastoma gene product (pRb) and on its ability to bind T-antigen were examined. Time course analysis showed no detectable inhibition of cellular protein and T-antigen synthesis up to 180 min treatment at 42.5 degrees C, while a gradual disappearance of the phosphorylated forms of pRb was observed after 90 min. Dephosphorylation of pRb was shown to correlate with inhibition of SV40-induced DNA synthesis. Also, large T-antigen/pRb protein complex was affected since a gradual dissociation of the hypophosphorylated pRb from large T-antigen was observed concomitant to the inhibition of DNA synthesis. The effects of heat treatment were found to be reversible after shifting the cultures to 37 degrees C. Cells were shown to resume DNA synthesis concomitant to the reappearance of the phosphorylated forms of pRb and rebinding of the hypophosphorylated form to large T-antigen within 6-8 h after recovery at 37 degrees C. In addition, no evidence for a bona fide hsp71/large T-antigen protein complex was seen under the experimental conditions used. The data strongly suggest that inhibition of DNA synthesis by heat treatment might involve either an increase in phosphatase activity or the inhibition of a pRb kinase activity.