The retinoblastoma tumor suppressor inhibits cellular proliferation through two distinct mechanisms: inhibition of cell cycle progression and induction of cell death

Abstract

Studies aimed at examining the precise function(s) of the retinoblastoma tumor suppressor protein, RB, have been hindered by the rapid phosphorylation and inactivation of ectopically expressed RB which occurs in the majority of cell types. Therefore, ectopically expressed RB is a poor inhibitor of cellular proliferation. We have designed constitutively active RB proteins, PSM-RB, that cannot be inactivated by phosphorylation. Using these proteins, we show that unlike wild-type RB, PSM-RB proteins inhibit cell cycle progression in a broad range of tumor cell types. Furthermore, unlike p16 ink4a , PSM-RB is also a potent inhibitor of cell cycle progression in RB-de®cient tumor cells. Surprisingly, we identi®ed a tumor cell line that is resistant to the cell cycle inhibitory e ects of PSM-RB. This ®nding challenges the hypothesis that RB must be inactivated in all cells for cell cycle progression to occur. Further characterization of this`resistant' tumor line revealed that proliferation of these cells is still inhibited by PSM-RB. We show that this is due to PSM-RB-induced cell death. As such, these studies are the ®rst to show that RB inhibits cellular proliferation through at least two distinct mechanisms ± inhibition of cell cycle progression and induction of cell death.Keywords: apoptosis; cyclin; phosphorylation; cell death; RB The retinoblastoma tumor suppressor protein, RB, is inactivated in over 60% of studied human tumors (Bartek et al., 1997;Sherr, 1996). Inactivation of RB can be achieved through multiple distinct mechanisms, including direct loss of functional protein due to mutation, binding of oncoproteins of DNA tumor viruses, or by overt phosphorylation of RB, which inactivates its growth-suppressing function (Bartek et al., 1997;Sherr, 1996;Wang et al., 1994). It has been hypothesized that RB must be phosphorylated and inactivated by cyclin dependent kinase (CDK)/cyclin complexes in cells to allow for cell cycle progression, and in tumor cells this process is often deregulated (Bartek et al., 1997;Sherr, 1996). For example, ampli®cation of the proto-oncogenes CDK4 or Cyclin D1 results in excessive phosphorylation and inactivation of RB. Likewise, loss of the tumor suppressor protein p16 ink4a which normally serves to attenuate the activity of CDK4 and cyclin D1, results in deregulated phosphorylation and inactivation of RB. It is thought that unphosphorylated (i.e. active) RB prevents cellular proliferation by sequestering a host of factors and assembling complexes which inhibit cell cycle progression (De Pinho, 1998;Sidle et al., 1996;Wang et al., 1994). Phosphorylation of RB by CDK/cyclin complexes causes release of these proteins, and thus enables cell cycle advancement.Studies aimed at directly de®ning the role of RB in cell cycle processes have been hindered by the fact that ectopically expressed wild-type RB is a very poor inhibitor of cellular proliferation, since it is phosphorylated and inactivated by endogenous CDK/Cyclin activity (Wang et al., 1994;Zacksenhaus et al., 1993). Because of ...

Cdk inhibitors, roscovitine and olomoucine, synergize with farnesyltransferase inhibitor (FTI) to induce efficient apoptosis of human cancer cell lines

“…Another possibility is that FTI in¯u-ences the activity of phosphatases that are responsible for regulating pRb phosphorylation status. Possible link between hypophosphorylation of pRb and apoptosis has recently been suggested (Dou et al, 1995;Knudsen et al, 1999). Ectopic expression of constitutively active form of pRb, which remains hypophosphorylated, induced apoptosis of cancer cells (Knudsen et al, 1999).…”

Anti-proliferative and pro-apoptotic activity of whole extract and isolated indicaxanthin from Opuntia ficus-indica associated with re-activation of the onco-suppressor p16INK4a gene in human colorectal carcinoma (Caco-2) cells

“…Besides being major controllers of the cell cycle, p16 INK4a and RB possess multiple antitumor functions [44] and their activity in regulating cell death has been shown. Over-expression of p16 INK4a has been associated with apoptosis of various transformed cells with down-regulation of the anti-apoptotic bcl-2 protein [45][46][47], and involvement of activated RB in apoptosis has been reported [48,49]. Early epigenetic changes during carcinogenesis can be induced, but also reverted, by external effectors, including food components.…”

Cyclin D1: polymorphism, aberrant splicing and cancer risk

“…Specifically, cyclin D1 has been shown to regulate a number of sequence-specific transcription factors, including C/EBPb (Lamb et al, 2003), STAT3 (Bienvenu et al, 2001), DMP1 (Inoue and Sherr, 1998), and BETA2/NeuroD (Ratineau et al, 2002). The largest class of transcription factors regulated by cyclin D1 belong to the nuclear receptor superfamily, and include the estrogen receptor (Zwijsen et al, 1998;Lamb et al, 2000), androgen receptor (Knudsen et al, 1999;Reutens et al, 2001;Petre et al, 2002;Burd et al, 2005), thyroid hormone receptor (Pibiri et al, 2001), and peroxisome proliferator activated receptor-g (Qin et al, 2003). In many cases cyclin D1 was shown to directly associate with the transcription factors, independent of CDK4 association, and modify transcription factor action through cell-cycle independent mechanisms.…”