Bey-Dih Chang

Role of p53 and p21waf1/cip1 in senescence-like terminal proliferation arrest induced in human tumor cells by chemotherapeutic drugs.

Exposure of human tumor cell lines to moderate doses of anticancer agents induces terminal proliferation arrest accompanied by morphologic and enzymatic changes that resemble senescence of normal cells. We have investigated the role of p53 and p21waf1/cip1 in the induction of this response in drug-treated tumor cells. Doxorubicin treatment induced the senescence-like phenotype (SLP) and its associated terminal growth arrest in wild-type HCT116 colon carcinoma cells; this response was strongly decreased but not abolished in HCT116 lines with homozygous knockout of p53 or p21. Transduction of HT1080 fibrosarcoma cells with a genetic inhibitor of p53 also decreased the induction of SLP and increased drug-induced mitotic cell death. To determine if drug-stimulated p21 expression was responsible for senescence-like growth arrest, we have expressed different levels of p21 from an inducible promoter. While high-level overexpression of p21 was sufficient to induce SLP in HT1080 cells, the levels of p21 expressed in doxorubicin-treated cells could account for only a fraction of doxorubicin-induced SLP. Our results indicate that p53 and p21 act as positive regulators of senescence-like terminal proliferation arrest, but their function is neither sufficient nor absolutely required for this treatment response in tumor cells.

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

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 p21Waf1/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.

p21Waf1/Cip1/Sdi1-induced growth arrest is associated with depletion of mitosis-control proteins and leads to abnormal mitosis and endoreduplication in recovering cells.

Induction of a cyclin-dependent kinase inhibitor p21Waf1/Cip1/Sdi1 is an integral part of cell growth arrest associated with senescence and damage response. p21 overexpression from an inducible promoter resulted in senescence-like growth arrest in a human fibrosarcoma cell line. After release from p21-induced growth arrest, cells re-entered the cell cycle but displayed growth retardation, cell death and decreased clonogenicity. The failure to form colonies was associated with abnormal mitosis and endoreduplication in the recovering cells and was correlated with the induced level of p21 and the duration of p21 induction. p21 induction was found to inhibit the expression of multiple proteins involved in the execution and control of mitosis. p21-induced depletion of the cellular pools of mitosis-control proteins was followed by asynchronous resynthesis of such proteins after release from p21, which explains the observed mitotic abnormalities. Genetic destabilization in cells recovering from p21-induced growth arrest may conceivably play a role in carcinogenesis and tumor progression.

Identification of potential anticancer drug targets through the selection of growth-inhibitory genetic suppressor elements

To identify human genes required for tumor cell growth, transcriptome-scale selection was used to isolate genetic suppressor elements (GSEs) inhibiting breast carcinoma cell growth. Growth-inhibitory GSEs (cDNA fragments that counteract their cognate gene) were selected from 57 genes, including known positive regulators of cell growth or carcinogenesis as well as genes that have not been previously implicated in cell proliferation. Many GSE-cognate genes encode transcription factors (such as STAT and AP-1) and signal transduction proteins. Monoclonal antibodies against a cell surface protein identified by GSE selection, neural cell adhesion molecule L1CAM, strongly inhibited the growth of several tumor cell lines but not of untransformed cells. Hence, selection for growth-inhibitory GSEs allows one to find potential targets for new anticancer drugs.

Cyclin-dependent kinase 8 mediates chemotherapy-induced tumor-promoting paracrine activities

Conventional chemotherapy not only kills tumor cells but also changes gene expression in treatment-damaged tissues, inducing production of multiple tumor-supporting secreted factors. This secretory phenotype was found here to be mediated in part by a damage-inducible cell-cycle inhibitor p21 (CDKN1A). We developed small-molecule compounds that inhibit damage-induced transcription downstream of p21. These compounds were identified as selective inhibitors of a transcription-regulating kinase CDK8 and its isoform CDK19. Remarkably, p21 was found to bind to CDK8 and stimulate its kinase activity. p21 and CDK8 also cooperate in the formation of internucleolar bodies, where both proteins accumulate. A CDK8 inhibitor suppresses damage-induced tumor-promoting paracrine activities of tumor cells and normal fibroblasts and reverses the increase in tumor engraftment and serum mitogenic activity in mice pretreated with a chemotherapeutic drug. The inhibitor also increases the efficacy of chemotherapy against xenografts formed by tumor cell/fibroblast mixtures. Microarray data analysis revealed striking correlations between CDK8 expression and poor survival in breast and ovarian cancers. CDK8 inhibition offers a promising approach to increasing the efficacy of cancer chemotherapy.

p21(Waf1/Cip1/Sdi1) mediates retinoblastoma protein degradation

Damage-induced G1 checkpoint in mammalian cells involves upregulation of p53, which activates transcription of p21Waf1 (CDKN1A). Inhibition of cyclin-dependent kinase (CDK)2 and CDK4/6 by p21 leads to dephosphorylation and activation of Rb. We now show that ectopic p21 expression in human HT1080 fibrosarcoma cells causes not only dephosphorylation but also depletion of Rb; this effect was p53-independent and susceptible to a proteasome inhibitor. CDK inhibitor p27 (CDKN1B) also caused Rb dephosphorylation and depletion, but another CDK inhibitor p16 (CDKN2A) induced only dephosphorylation but not depletion of Rb. Rb depletion was observed in both HT1080 and HCT116 colon carcinoma cells, where p21 was induced by DNA-damaging agents. Rb depletion after DNA damage did not occur in the absence of p21, and it was reduced when p21 induction was inhibited by p21-targeting short hairpin RNA or by a transdominant inhibitor of p53. These results indicate that p21 both activates Rb through dephosphorylation and inactivates it through degradation, suggesting negative feedback regulation of damage-induced cell-cycle checkpoint arrest.

Identification of Promoter Elements Responsible for Transcriptional Inhibition of Polo-like Kinase 1 and Topoisomerase IIα Genes by p21WAF1/CIP1/ SDI1

Induction of p21WAF1/CIP1/SDI1, a physiological mediator of cell cycle arrest, inhibits multiple genes involved in cell division. We have investigated the determinants of p21-mediated inhibition of two of these genes, polo-like kinase 1 (PLK1) and topoisomerase IIa (TOPO IIa). p21 expression from an inducible promoter in human HT1080 cells rapidly decreases cellular levels of PLK1 and TOPO IIa RNA without decreasing their RNA stability. p21 also inhibits reporter gene expression from the PLK1 and TOPO IIa promoters in transient and stable transfection assays. Promoter mutagenesis studies show that inhibition of the PLK1 promoter by p21 is mediated in part by tandem sequences CDE (cell cycle-dependent element) and CHR (cell cycle genes homology region). p21 response of the TOPO IIa promoter was found to be mediated through CDE (but not CHR) and the inverted CCAAT box 1 (ICB1). The extent of PLK1 and TOPO IIa promoter inhibition and the effects of promoter mutations differ under the conditions of growth arrest produced by p21 induction or by mimosine, a cell cycle inhibitor that increases p21 RNA but not protein expression in HT1080 cells. These results indicate that inhibition of cell division-associated genes by p21 is mediated by different but overlapping mechanisms, which are not a general consequence of cell cycle arrest. Key Words p21WAF1/CIP1/SDI1, Polo-like Kinase, Topoisomerase II, Regulation of Transcription, CDE, CHR, ICB

Retinoid-Induced Growth Arrest of Breast Carcinoma Cells Involves Co-Activation of Multiple Growth-Inhibitory Genes

Retinoids are used in leukemia therapy and chemoprevention of cancers. Treatment of MCF-7 breast carcinoma cells with low doses of retinoids induces gradual proliferation arrest with phenotypic markers of senescence. cDNA microarray hybridization and reverse transcription-polymerase chain reaction analysis showed that retinoid-induced growth arrest in MCF-7 cells is associated with strong induction of 13 genes. Four of these genes (IGF-binding protein 3, EPLIN, βIG-H3 and FAT10) have antiproliferative activity; EPLIN and βIG-H3 are also known to be selectively inhibited in transformed relative to normal cells. The functions of the induced genes may also account for other cellular effects of retinoids, including the proteasome-mediated protein degradation, increased cell adhesion, and retinoic acid synthesis. Only one of 13 strongly induced genes (ring finger protein TRIM31) contains a putative retinoid response element in its promoter; TRIM31 also shows the most rapid kinetics of induction by retinoids. In contrast, the antiproliferative genes contain no identifiable retinoid response elements in their promoters and show more gradual induction kinetics, suggesting that these genes are indirectly induced by retinoids. Elucidation of the mechanisms that mediate co-induction of growth-inhibitory genes in retinoid-treated cells may suggest an approach to reproducing the growth-inhibitory effect of retinoids in retinoid-insensitive human cancers.

Applications of green fluorescent protein as a marker of retroviral vectors

Abstracthe Green Fluorescent Protein (GFP) ofAequorea victoria is used as a vital fluorescent tag for the detection and isolation of genetically modified cells. Several modified variants of GFP were tested as marker genes in retroviral vectors containing different backbones and promoter combinations. Constructs allowing for reliable detection of GFP fluorescence and the expression of a cotransduced gene from a strong promoter were identified. Cells harboring such constructs are detectable by flow cytometry, fluorescence microscopy and multi-well fluorescence reading. GFP expression in transduced cells is stable both in vitro and in vivo, and long-term dynamics of GFP-positive fractions in a mixed population can be used to monitor the biological effects of a cotransduced gene. Selection of cells with the highest GFP fluorescence enriches for multiply infected cells. The use of different GFP variants allows one to monitor simultaneously two cell populations transduced with vectors carrying GFPs that differ in their fluoesscence intensity or spectral properties and to identify doubly transduced cells. In addition, transcription of an inducible promoter positioned in the opposite orientation to GFP can be monitored by the inhibition of GFP fluorescence. Thus, GFP provides a useful marker for gene transfer by retroviral vectors and extends the range of applications for retroviral transduction.

Inducible retroviral vectors regulated by lac repressor in mammalian cells.

We have developed lac repressor-regulated retroviral expression vectors that are induced by beta-galactosides upon stable transduction in mammalian cells. These vectors, derived from a Moloney-virus-based vector LNCX, contain an internal Rous sarcoma virus (RSV) or cytomegalovirus (CMV) promoter, coupled with 2 to 4 lac operator sequences and placed in anti orientation relative to the retroviral long terminal repeat (LTR). Three different vectors were tested in stably infected mass populations of mouse and human cells expressing the lac repressor, in parallel with the constitutively expressed LNCX vector. The highest expression levels from these vectors ranged from 1-4% to 25-33% of the LNCX level, and the induction by beta-galactosides ranged from 6-11-fold to 29-54-fold. These vectors should be suitable for studies requiring efficient gene transfer and regulated expression in mass populations of stably transduced mammalian cells.