Hyperphosphorylation Of Retinoblastoma Protein Research Articles

Inhibition of G1 Cyclin Expression in Normal Rat Kidney Cells by Inostamycin, a Phosphatidylinositol Synthesis Inhibitor

We previously reported that inostamycin, an inhibitor of CDP-DG: inositol transferase, inhibited cell proliferation in normal rat kidney (NRK) cells by blocking cell cycle progression at the G1 phase. In the present paper, we report the effect of inostamycin on the serum-induced activation of Ser/Thr protein kinases that are involved in G1 progression. In quiescent NRK cells mitogen-activated protein kinase (MAP kinase) and casein kinase II were activated within 15 min after serum addition. Neither activation was affected by the treatment with inostamycin. However, in the inostamycin-treated cell, cyclin-dependent kinase 2 (CDK2) failed to be activated after serum stimulation. Since serum-induced expression of cyclin E was also suppressed by inostamycin, this inhibitor would appear to block CDK2 activation by inhibiting cyclin E expression. Furthermore, inostamycin also inhibited cyclin D1 expression induced by serum; and consequently, hyperphosphorylation of retinoblastoma protein (pRB) by RB-kinases such as CDK4 and CDK2 was abolished, which would result in elimination of functional inactivation of pRB. Thus, early G1 arrest in NRK cells by inostamycin is due to the inhibition of cyclin D1 and E expressions.

Hyperphosphorylation of retinoblastoma protein and stimulation of growth by okadaic acid in human pancreatic cancer.

Phosphorylation/dephosphorylation of intracellular proteins are important steps in the regulation of cell growth. Okadaic acid, an inhibitor of the serine/threonine protein phosphatases 1 and 2A, is a potent tumor promoter. This effect may be through the inhibition of dephosphorylation (termed "hyperphosphorylation") and subsequent inactivation of tumor-suppressor proteins. We examined whether okadaic acid regulates growth of human pancreatic cancer cells (MIA PaCa-2 and Panc-1) or alters the phosphorylation of the retinoblastoma tumor-suppressor protein. Growth studies, nuclear labeling analyses, and Western blotting for retinoblastoma protein were performed. Okadaic acid stimulated cell growth and induced hyperphosphorylation of the retinoblastoma protein. The growth-stimulatory effect of okadaic acid on these human pancreatic cancer cells may be mediated by inactivation of the growth suppressive effect of the retinoblastoma protein by hyperphosphorylation. These studies suggest that the growth of these human pancreatic cancer cells is still regulated by tumor-suppressor proteins.

K252a Inhibits the Phosphorylation of pRb without Changing the Levels of G1 Cyclins and Cdk2 Protein in Human Hepatoma Cells

A protein kinase inhibitor K252a suppressed the growth of HuH7 hepatoma cells and the hyperphosphorylation of retinoblastoma protein (pRb) at late G1phase of cell cycle. However, K252a treatment did not alter the levels of cyclin D1, cyclin E, cyclin A and Cdk2 protein bound to cyclin E or cyclin A. Therefore, the K252a inhibition of pRb phosphorylation is considered to be brought about probably by inhibiting the action of Cdk-cyclin complex rather than by changing its cellular level. These results also suggest that K252a is a useful tool for investigating the mechanism of phosphorylation of pRb mediated by Cdk-cyclin.

Retinoblastoma protein in non-small cell lung carcinoma, cells arrested for growth by retinoic acid: Maxwell SA. Section Thoracic Molecular Oncology, Dept Thoracic Cardiovascular Surgery, Univ Texas MD Anderson Cancer Center, Houston, TX. Anticancer Res 1994; 14: 1535–1540

The growth of non-small cell lung carcinoma (NSCLC) cells was inhibited by retinoic acid after 16 h of treatment. However, the growth of all cell lines except one became refractory to retinoic acid after 48 h of exposure. The expression of the hyperphosphorylated retinoblastoma protein species (RB p110) was observed to be increased fivefold to tenfold in NSCLC cells within 16 h of exposure to retinoic acid. In the H460a and H226b cell lines, p110 showed some conversion to the underphosphorylated p105 form after 24 h of retinoic acid treatment. After 48 h, p105 became the predominant form, along with a 60K M(r) species. After 72 h, expression of all RB protein species became almost undetectable in H460a and H226b cells, concomitant with increases in cell growth rates. A different pattern of RB expression was observed in the H322j and H358 cell lines. In these cells, both p110 and p105 were induced within 8 h of retinoic acid treatment. Furthermore, the elevated levels and the phosphorylation state of RB in retinoic acid-treated H322j and H358 cells remained essentially unchanged for up to 72 h and only low amounts of the 60K M(r) species were detected. I believe that a post-translational mechanism is responsible for the retinoic acid-mediated induction of RB, since levels of RB mRNA remained relatively unchanged during the time course of retinoic acid exposure. I conclude that retinoic acid inhibited the growth of NSCLC cells by inducing high levels of RB and that increases in RB levels occurred as a result of either an increase in stability of the protein or by downregulation of an RB-specific protease. The inhibition of growth by retinoic acid must involve other molecular events, since the H596b cell line, which lacks RB protein, exhibited growth properties in the presence of retinoic acid similar to those of cells in which RB expression was induced.

Transforming growth factor beta effects on expression of G1 cyclins and cyclin-dependent protein kinases.

Transforming growth factor beta 1 (TGF-beta 1) is a potent growth-inhibitory polypeptide. The mechanism of TGF-beta 1 inhibition has been related to its ability to prevent the hyperphosphorylation of retinoblastoma protein (pRb). Several lines of evidence have suggested that cell cycle-regulated protein kinases are responsible for the hyperphosphorylation of pRb. We demonstrate here that TGF-beta 1 has profound effects on the expression of genes encoding certain G1 cyclins and their associated kinases, which provides one explanation of TGF-beta 1 effects on pRb hyperphosphorylation. These results also suggest that the growth-inhibitory effects of TGF-beta 1 in many cells are attributable to its effects on the cell cycle apparatus involved in programming G1 transit.