Deficiency of G1 regulators P53, P21Cip1 and/or pRb decreases hepatocyte sensitivity to TGFβ cell cycle arrest

Background: TGF beta is critical to control hepatocyte proliferation by inducing G1-growth arrest through multiple pathways leading to inhibition of E2F transcription activity. The retinoblastoma protein pRb is a key controller of E2F activity and G1/S transition which can be inhibited in viral hepatitis. It is not known whether the impairment of pRb would alter the growth inhibitory potential of TGF beta in disease. We asked how Rb-deficiency would affect responses to TGF beta-induced cell cycle arrest. Results: Primary hepatocytes isolated from Rb-floxed mice were infected with an adenovirus expressing CRE-recombinase to delete the Rb gene. In control cells treatment with TGF beta prevented cells to enter S phase via decreased cMYC activity, activation of P16(INK4A) and P21(Cip) and reduction of E2F activity. In Rb-null hepatocytes, cMYC activity decreased slightly but P16(INK4A) was not activated and the great majority of cells continued cycling. Rb is therefore central to TGF beta-induced cell cycle arrest in hepatocytes. However some Rb-null hepatocytes remained sensitive to TGF beta-induced cell cycle arrest. As these hepatocytes expressed very high levels of P21(Cip1) and P53 we investigated whether these proteins regulate pRb-independent signaling to cell cycle arrest by evaluating the consequences of disruption of p53 and p21(Cip1). Hepatocytes deficient in p53 or p21(Cip1) showed diminished growth inhibition by TGF beta. Double deficiency had a similar impact showing that in cells containing functional pRb; P21(Cip) and P53 work through the same pathway to regulate G1/S in response to TGF beta. In Rb-deficient cells however, p53 but not p21(Cip) deficiency had an additive effect highlighting a pRb-independent- P53-dependent effector pathway of inhibition of E2F activity. Conclusion: The present results show that otherwise genetically normal hepatocytes with disabled p53, p21(Cip1) or Rb genes respond less well to the antiproliferative effects of TGF beta. As the function of these critical cellular proteins can be impaired by common causes of chronic liver disease and HCC, including viral hepatitis B and C proteins, we suggest that disruption of pRb function, and to a lesser extend P21(Cip1) and P53 in hepatocytes may represent an additional new mechanism of escape from TGF beta-growth-inhibition in the inflammatory milieu of chronic liver disease and contribute to cancer development.