OVEREXPRESSION OF HUMAN P21­­ʷᵃᶠ¹⸍ᶜⁱᵖ¹ ARRESTS THE GROWTH OF CHICKEN EMBRYO FIBROBLASTS TRANSFORMED BY INDIVIDUAL ONCOGENES

Abstract

In normal cells, cell growth and division are controlled by the interplay between proto-oncogenes and tumor suppressor genes. Cancer cells usually have both activated an oncogene and lost a functional tumor suppressor gene. This study addresses the interrelationship between these positive and negative growth regulators. The main focus is on p21­­ʷᵃᶠ¹⸍ᶜⁱᵖ¹, a cyclin/cdk inhibitor whose expression is regulated by p53. High level expression of the p53 tumor suppressor can block the growth of cancer cells. Wafl/cipl is transactivated by p53 and the p21ʷᵃᶠ¹⸍ᶜⁱᵖ¹ protein is itself a suppressor of cell growth. To test the growth suppression effect of p21ʷᵃᶠ¹⸍ᶜⁱᵖ¹ or p53 on the growth of normal cells and cells transformed by individual oncogenes, we used replication-competent retroviral vectors to induce high level expression of p53 and p21ʷᵃᶠ¹⸍ᶜⁱᵖ¹ in chicken embryo fibroblasts (CEF). These are primary cells and by using them we avoid the complications that arise when such experiments are done with established cell lines. This study shows that overexpression of p21ʷᵃᶠ¹⸍ᶜⁱᵖ¹ or p53 arrests the growth of CEF at the G 1 phase of the cell cycle by inhibiting DNA synthesis. We next asked whether the growth inhibition induced by p21ʷᵃᶠ¹⸍ᶜⁱᵖ¹ could be overcome by mitogenic signals delivered by a variety of oncogenes. We show that the growth of CEF transformed by v-Src, tf-Ras, c-Mos and c-Myc is inhibited by overexpression of p21ʷᵃᶠ¹⸍ᶜⁱᵖ¹. This suggests that p21ʷᵃᶠ¹⸍ᶜⁱᵖ¹ functions downstream of these oncogenes, suggesting that mitogenic signals converge at the cyclin/cdk complex. To address the interplay between positive and negative signals within the cell cycle machinery, we used this system to test the effects of overexpressing both p21ʷᵃᶠ¹⸍ᶜⁱᵖ¹ and E2F1, a subunit of the E2F transcription factor, on the growth of CEF. The E2F transcription factors are released from binding to hypophosphorylated pRb when pRb is phosphorylated by cyclin/cdks. This allows for transcription of S phase genes and progression from G₁ to S phase in the cell cycle. In this system very high levels of E2F1 overexpression cause considerable apoptosis, however the surviving cells still overexpress E2F1. These cells are transformed and their growth is blocked by overexpression of p21ʷᵃᶠ¹⸍ᶜⁱᵖ¹. These data suggest that p21ʷᵃᶠ¹⸍ᶜⁱᵖ¹ might be useful for gene therapy for human cancer. We also show that the apoptosis induced by high levels of E2F1 can be blocked by Bc1-2, an inhibitor of apoptosis.