Molecular determinants of terminal growth arrest induced in tumor cells by a chemotherapeutic agent

Abstract

Treatment with chemotherapy or radiation is not invariably cytotoxic to all tumor cells. Some of the cells that survive treatment recover and resume proliferation, whereas others undergo permanent growth arrest. To understand the nature of treatment-induced terminal growth arrest, colon carcinoma cells were exposed to doxorubicin, and surviving cells were separated into proliferating and growth-arrested populations. Only growth-arrested cells displayed phenotypic markers of cell senescence and failed to form colonies. Gene expression was compared between senescent and proliferating fractions of drug-treated cells by using cDNA microarray hybridization and reverse transcription-PCR. Drug-induced senescence was associated with inhibition of genes involved in cell proliferation and with coinduction of multiple intracellular and secreted growth inhibitors. Several tumor suppressors and other genes that are down-regulated in carcinogenesis were up-regulated in senescent tumor cells. Induction of most growth inhibitors was delayed but not abolished in cells with homozygous knockout of p53, in agreement with only limited p53 dependence of drug-induced terminal growth arrest. On the other hand, senescent cells overexpressed secreted proteins with antiapoptotic, mitogenic, and angiogenic activities, suggesting that drug-induced senescence is associated with paracrine tumor-promoting effects. About one-third of the genes up-regulated in senescent cells and almost all of the down-regulated genes showed decreased or delayed changes in p21(Waf1/Cip1/Sdi1)-deficient cells, indicating that p21 is a major mediator of the effects of p53 on gene expression. Elucidation of molecular changes in tumor cells that undergo drug-induced senescence suggests potential strategies for diagnostics and therapeutic modulation of this antiproliferative response in cancer treatment.

Figure 1

Separation and analysis of proliferating and growth-arrested fractions of doxorubicin-treated HCT116 cells. (A) FACS profiles of PKH2 fluorescence of HCT116 cells on the indicated days after release from doxorubicin. PKH2^lo population of proliferating cells appears on day 4 and separates from the PKH2^hi (growth-arrested) population by day 6. (B) FACS analysis of DNA content of exponentially growing HCT116 cells (green) and of PKH2^hi population isolated 9 days after drug treatment (red). (C) SA-β-gal staining of PKH2^hi (Right) and PKH2^lo (Left) populations, separated 6 days after release from the drug. Both Left and Right are photographed at the ×200. (D) Colony formation by PKH2^hi and PKH2^lo populations, separated 9 days after drug treatment and plated at 10,000 live (propidium iodide-negative) cells per 10-cm plate.

Figure 2

RT-PCR analysis of changes in the expression of the indicated senescence-associated genes. β-actin was used as a normalization standard. (A) Gene expression in proliferating (PKH2^lo) and senescent (PKH2^hi) populations of HCT116 cells, separated 9 days after doxorubicin treatment. (B) Gene expression in the unsorted populations of wild-type, p21−/−, and p53−/− HCT116 cells, before and after 24-hr treatment with 200 nM doxorubicin, and on the indicated days after release from the drug. Genes were designated as p53- or p21-dependent if changes in their expression became detectable at a later day or were less pronounced in the p53−/− or p21−/− lines than in the wild-type cells.

Figure 3

Immunoblotting analysis of changes in p53 and the indicated protein products of genes that show altered expression in drug-induced senescence. β-actin was used as a normalization standard. (A) Immunoblotting of wild-type HCT116 cells that were either untreated, treated for 2 days with 200 nM doxorubicin, or sorted into proliferating (PKH2^lo) and senescent (PKH2^hi) cell populations 9 days after doxorubicin treatment. (B) p53 dependence of p21 induction in doxorubicin-treated HCT116 cells. Immunoblotting analysis of the wild-type, p21−/−, and p53−/− HCT116 cell lines treated with doxorubicin for the indicated number of days.