Genome Editing As an Approach to the Study of in Vivo Transcription Reprogramming.

CDK8-mediated transcriptional reprogramming is essential for an extensive gene expression. Constitutive knockouts of the cdk8 gene are lethal at the morula stage. For modeling transcriptional reprogramming in an adult organism, we investigated the possibility to attenuate the CDK8 kinase activity with a F97G mutation in exon 3 of the cdk8 gene. According to preliminary experimental data, this mutation should lead to a decrease in CDK8 kinase activity. To edit the genome of laboratory mice, the CRISPR/Cas9 technology was used, in which the introduction of a double-stranded gap occurred at a distance of 128 nucleotide pairs from the planned site of the introduced mutation. To introduce the mutation, a matrix for homologous repair was used as part of plasmid DNA, with homologous arms 903 and 484 bp in the 5'-3' region from the point of double-stranded rupture, respectively. As a result, mice with site-specific target mutations in exon 3 of the cdk8 gene were obtained. We for the first time demonstrated a high efficacy of the mutation 128 bp apart from the site of double-strand break. Viable animals with the F97G mutation in the catalytic domain of CDK8 kinase were obtained for the first time. The resulting cdk8 mutant mice will be used in subsequent studies to simulate the processes involving transcription reprogramming.

Cellular senescence induced by aberrant MAD2 levels impacts on paclitaxel responsiveness in vitro.

BACKGROUND: The mitotic arrest deficiency protein 2 (MAD2) is a key component of the mitotic spindle assembly checkpoint, monitoring accurate chromosomal alignment at the metaphase plate before mitosis. MAD2 also has a function in cellular senescence and in a cell's response to microtubule inhibitory (MI) chemotherapy exemplified by paclitaxel. METHODS: Using an siRNA approach, the impact of MAD2 down-regulation on cellular senescence and paclitaxel responsiveness was investigated. The endpoints of senescence, cell viability, migration, cytokine expression, cell cycle analysis and anaphase bridge scoring were carried out using standard approaches. RESULTS: We show that MAD2 down-regulation induces premature senescence in the MCF7 breast epithelial cancer cell line. These MAD2-depleted (MAD2) cells are also significantly replicative incompetent but retain viability. Moreover, they show significantly higher levels of anaphase bridges and polyploidy compared to controls. In addition, these cells secrete higher levels of IL-6 and IL-8 representing key components of the senescence-associated secretory phenotype (SASP) with the ability to impact on neighbouring cells. In support of this, MAD2 cells show enhanced migratory ability. At 72 h after paclitaxel, MAD2 cells show a significant further induction of senescence compared with paclitaxel naive controls. In addition, there are significantly more viable cells in the MAD2 MCF7 cell line after paclitaxel reflecting the observed increase in senescence. CONCLUSION: Considering that paclitaxel targets actively dividing cells, these senescent cells will evade cytotoxic kill. In conclusion, compromised MAD2 levels induce a population of senescent cells resistant to paclitaxel.

Human multidrug-resistant cell lines: increased mdr1 expression can precede gene amplification.

The development of simultaneous resistance to multiple structurally unrelated drugs is a major impediment to cancer chemotherapy. Multidrug resistance in human KB carcinoma cells selected in colchicine, vinblastine, or Adriamycin is associated with amplification of specific DNA sequences (the multidrug resistance locus, mdr1). During colchicine selection resistance is initially accompanied by elevated expression of a 4.5-kilobase mdr1 messenger RNA (mRNA) without amplification of the corresponding genomic sequences. During selection for increased levels of resistance, expression of this mRNA is increased simultaneously with amplification of mdr1 DNA. Increased expression and amplification of mdr1 sequences were also found in multidrug-resistant sublines of human leukemia and ovarian carcinoma cells. These results suggest that increased expression of mdr1 mRNA is a common mechanism for multidrug resistance in human cells. Activation of the mdr1 gene by mutations or epigenetic changes may precede its amplification during the development of resistance.

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

Damage-induced G1 checkpoint in mammalian cells involves upregulation of p53, which activates transcription of p21(Waf1) (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.

Decreased expression of the amplified mdr1 gene in revertants of multidrug-resistant human myelogenous leukemia K562 occurs without loss of amplified DNA.

Amplification and increased expression of the mdr1 gene associated with multidrug resistance in human tumors were found in multidrug-resistant sublines of human myelogenous leukemia K562 selected with vincristine (K562/VCR) or adriamycin (K562/ADM). In two revertant cell lines of K562/ADM, amplification of the mdr1 gene was maintained at the same level as in K562/ADM, but expression of the 4.5-kilobase mdr1 mRNA was greatly decreased, indicating that amplified genes may be inactivated at the level of transcription without a corresponding loss of amplified DNA.

Structure and expression of the human MDR (P-glycoprotein) gene family.

The human MDR (P-glycoprotein) gene family is known to include two members, MDR1 and MDR2. The product of the MDR1 gene, which is responsible for resistance to different cytotoxic drugs (multidrug resistance), appears to serve as an energy-dependent efflux pump for various lipophilic compounds. The function of the MDR2 gene remains unknown. We have examined the structure of the human MDR gene family by Southern hybridization of DNA from different multidrug-resistant cell lines with subfragments of MDR1 cDNA and by cloning and sequencing of genomic fragments. We have found no evidence for any other cross-hybridizing MDR genes. The sequence of two exons of the MDR2 gene was determined from genomic clones. Hybridization with single-exon probes showed that the human MDR1 gene is closely related to two genes in mouse and hamster DNA, whereas MDR2 corresponds to one rodent gene. The human MDR locus was mapped by field-inversion gel electrophoresis, and both MDR genes were found to be linked within 330 kilobases. The expression patterns of the human MDR genes were examined by enzymatic amplification of cDNA. In multidrug-resistant cell lines, increased expression of MDR1 mRNA was paralleled by a smaller increase in the levels of MDR2 mRNA. In normal human tissues, MDR2 was coexpressed with MDR1 in the liver, kidney, adrenal gland, and spleen. MDR1 expression was also detected in colon, lung, stomach, esophagus, muscle, breast, and bladder.

Multidrug resistance of DNA-mediated transformants is linked to transfer of the human mdr1 gene.

Mouse NIH 3T3 cells were transformed to multidrug resistance with high-molecular-weight DNA from multidrug-resistant human KB carcinoma cells. The patterns of cross resistance to colchicine, vinblastine, and doxorubicin hydrochloride (Adriamycin; Adria Laboratories Inc.) of the human donor cell line and mouse recipients were similar. The multidrug-resistant human donor cell line contains amplified sequences of the mdr1 gene which are expressed at high levels. Both primary and secondary NIH 3T3 transformants contained and expressed these amplified human mdr1 sequences. Amplification and expression of the human mdr1 sequences and amplification of cotransferred human Alu sequences in the mouse cells correlated with the degree of multidrug resistance. These data suggest that the mdr1 gene is likely to be responsible for multidrug resistance in cultured cells.

Autonomously replicating episomes contain mdr1 genes in a multidrug-resistant human cell line.

Gene amplification in human tumor cells is frequently mediated by extrachromosomal elements (e.g., double minute chromosomes [DMs]). Recent experiments have shown that DMs can be formed from smaller, submicroscopic circular precursors referred to as episomes (S. M. Carroll, M. L. DeRose, P. Gaudray, C. M. Moore, D. R. Needham-Vandevanter, D. D. Von Hoff and G. M. Wahl, Mol. Biol. 8:1525-1533, 1988). To investigate whether episomes are generally involved as intermediates in gene amplification, we determined whether they mediate the amplification of the mdr1 gene, which when overexpressed engenders cross resistance to multiple lipophilic drugs. A variety of methods including electrophoresis of undigested DNAs in high-voltage gradients, NotI digestion, and production of double-strand breaks by gamma irradiation were used to distinguish between mdr1 sequences amplified on submicroscopic circular molecules and those amplified within DMs or chromosomal DNA. The gamma-irradiation procedure provides a new method for detecting and determining the size of circular molecules from 50 kilobases (kb) to greater than 1,000 kb. These methods revealed that some of the amplified mdr1 genes in vinblastine-resistant KB-V1 cells are contained in supercoiled circular molecules of approximately 600 and approximately 750 kb. Analysis of the replication of these molecules by a Meselson-Stahl density shift experiment demonstrated that they replicate approximately once in a cell cycle. The data lend further support to a model for gene amplification in which DMs are generally formed from smaller, autonomously replicating precursors.

Induction of multidrug resistance in human cells by transient exposure to different chemotherapeutic drugs.

BACKGROUND: The MDR1 (multidrug resistance) gene (also known as PGY1), which encodes the transmembrane efflux pump P-glycoprotein (Pgp), confers resistance to Pgp-transported cytotoxic drugs in tissue culture. The increase in MDR1 expression in tumors after chemotherapy is usually attributed to selection of pre-existing multidrug-resistant cells by Pgp-transported drugs. MDR1 expression in tissue culture can be increased by several types of stress-inducing treatment, including agents that activate protein kinase C (PKC). Previous studies, however, failed to demonstrate that short-term exposure to any chemotherapeutic drug can induce the expression of the resident MDR1 gene in drug-sensitive human cells. PURPOSE: This study was designed to utilize highly sensitive assays to determine if transient exposure to chemotherapeutic drugs would have an effect on MDR1 expression in human cells and to assess if PKC inhibitors would influence such an effect. METHODS: We analyzed the MDR1 gene expression in several human cell lines derived from leukemias or solid tumors, after treatment with different cytotoxic drugs, in the presence or absence of PKC inhibitors. Pgp function and expression were studied by flow cytometric assays, and MDR1 messenger RNA (mRNA) was assayed by polymerase chain reaction. RESULTS: Transient exposure to chemotherapeutic drugs, including agents that are not transported by Pgp, induced Pgp and MDR1 mRNA expression in subpopulations of treated cells in most of the tested cell lines. This induction was observed along with microscopically detectable cell damage. The drug-induced MDR1 expression and the associated twofold to threefold increase in resistance to vinblastine were sustained in K562 leukemia cells for at least several weeks after the removal of the drug. Drug-mediated MDR1 induction was blocked by nonspecific protein kinase inhibitors that are active against PKC, but not by a protein kinase inhibitor ineffective against PKC. CONCLUSIONS: Expression of the human MDR1 mRNA and Pgp can be induced in response to cellular damage by cytotoxic drugs regardless of whether the drugs are transported by Pgp. This induction can be prevented by protein kinase inhibitors. IMPLICATIONS: Induction of MDR1 expression in response to cellular damage may account for increased MDR1 expression in treated human tumors. Protein kinase inhibitors may be useful in preventing the emergence of multidrug resistance during cancer chemotherapy.

Clinical correlates of MDR1 (P-glycoprotein) gene expression in ovarian and small-cell lung carcinomas.

BACKGROUND: Expression of the MDR1 (P-glycoprotein) gene causes resistance to several classes of lipophilic anti-cancer drugs, but MDR1 expression in untreated ovarian and lung carcinomas is rarely detectable by standard assays. PURPOSE: This study was designed to measure the MDR1 messenger RNA (mRNA) content of ovarian and lung carcinomas and to analyze clinical correlations of MDR1 expression in these tumors. METHODS: A sensitive assay based on the polymerase chain reaction (PCR) was used in a retrospective study to measure MDR1 mRNA in biopsy samples of 100 solid tumors, including 60 ovarian and 32 lung carcinomas. The levels of MDR1 mRNA were correlated with history of chemotherapeutic treatment for all tumors; for ovarian and small-cell lung carcinomas (SCLCs), these levels were also correlated with subsequent tumor response to chemotherapy. RESULTS: Among previously untreated patients, MDR1 mRNA was expressed in 68% (50 of 74) of all tumors. Among patients pretreated with chemotherapy regimens that included at least one P-glycoprotein-transported drug (MDR regimens), 95% (20 of 21) of all tumors expressed MDR1 mRNA though the incidence of high-level MDR1 expression was decreased among the treated tumors. MDR1 mRNA was expressed in only one of five tumors treated with regimens that included no P-glycoprotein substrates (non-MDR regimens). Subsequent tumor response to chemotherapy was evaluated in 35 patients with ovarian carcinoma and seven patients with SCLC. The presence of even very low levels of MDR1 mRNA correlated with the lack of response to MDR regimens in these tumor types (P < .035 for ovarian carcinomas, P < .029 for SCLCs, and P < .0005 for both tumor types; Fisher's Exact Test). CONCLUSIONS: Low-level expression of MDR1 mRNA correlates with clinical resistance to combination chemotherapy in ovarian cancer and SCLC. We hypothesize that MDR1 is expressed in a subpopulation of more malignant tumor cells possessing multiple mechanisms of drug resistance. IMPLICATIONS: The presence of MDR1-expressing tumor cells may be useful as a predictive marker for clinical resistance to combination chemotherapy in ovarian cancer and SCLC. Prospective studies are needed to confirm this hypothesis.

Effects of the multidrug transporter P-glycoprotein on cellular responses to ionizing radiation.

Ionizing radiation induces apoptosis, mitotic catastrophe, and senescence-like terminal proliferation arrest in tumor cells. We investigated the effect of the MDR1 P-glycoprotein (Pgp), recently shown to inhibit caspase-mediated apoptosis, on cellular responses to radiation. Pgp strongly inhibited radiation-induced apoptosis in a HeLa-derived cell line with inducible MDR1 expression and in NIH 3T3 cells transduced with a MDR1-expressing retroviral vector. The inhibition of apoptosis by Pgp was associated, however, with increases in radiation-induced mitotic catastrophe and senescence and produced only a marginal change in the survival of irradiated cells. Pgp had no effect on radiation responses in apoptosis-resistant HT1080 cells. These results indicate that Pgp inhibits radiation-induced apoptosis, but this effect of Pgp provides no substantial increase in radiation resistance of the tested cell lines because apoptosis-resistant cells die from mitotic catastrophe or undergo senescence-like terminal proliferation arrest.

Pleiotropic resistance to DNA-interactive drugs is associated with increased expression of genes involved in DNA replication, repair, and stress response.

A combination of four genetic suppressor elements (GSEs), two of which are derived from putative transcriptional regulators, was previously found to increase resistance to drugs inhibiting DNA replication in HT1080 fibrosarcoma cells. In the present study, two GSE-transduced cell lines, isolated with and without cytotoxic selection, were found to be resistant to a diverse group of DNA-interactive agents, including aphidicolin, hydroxyurea, cytarabine, etoposide, doxorubicin, and mafosfamide. Changes in gene expression associated with GSE-induced drug resistance were analyzed by cDNA array hybridization and reverse transcription-PCR. Twenty genes were found to be up-regulated in both of the resistant cell lines. These include genes involved in DNA replication and repair (e.g., PCNA, XRCC1, B-MYB, and GADD45), transcriptional regulators associated with stress response, and cell cycle checkpoint control (e.g., YB-1, DBPA, and ATF4), and genes for signal transduction proteins (e.g., protein tyrosine phosphatase 1B and regulatory subunits alpha and beta of cAMP-dependent protein kinase). The observed changes in gene expression may play a role in pleiotropic resistance to different classes of DNA-targeting drugs.

Altered expression of ubiquitous kinesin heavy chain results in resistance to etoposide and hypersensitivity to colchicine: mapping of the domain associated with drug response.

The motor protein kinesin is a tetramer consisting of two heavy and two light chains. Expression of an antisense RNA fragment derived from the mouse ubiquitous kinesin heavy chain (uKHC) cDNA is associated with a unique type of multidrug resistance. We analyzed the effects of retroviral transduction of the human uKHC and its derivatives on drug sensitivity of the human fibrosarcoma cell line HT1080. Surprisingly, overexpression of full-length uKHC and its variants that were deficient in the NH2-terminal motor domain produced a phenotype similar to that of antisense RNA, characterized by resistance to etoposide and collateral sensitivity to colchicine. This altered drug response, therefore, appears to be a general consequence of kinesin deregulation. The genetic suppressor element approach was applied to map the determinants of drug response in the kinesin heavy chain. A sense-oriented genetic suppressor element conferring resistance to etoposide was isolated from a retroviral library of randomly fragmented uKHC cDNA. This element encodes the last 55 amino acids of uKHC, suggesting that the COOH-terminal tail domain of uKHC is involved in the cellular drug response.

Association of Ki-ras with amplified DNA sequences, detected in human ovarian carcinomas by a modified in-gel renaturation assay.

A modified in-gel DNA renaturation technique, which detects DNA sequences amplified greater than 7-fold in human DNA, was used to analyze gene amplification in surgical specimens of primary and metastatic ovarian carcinomas. Amplified DNA sequences were detected in two of eight tumors. Hybridization of these samples with different oncogene probes revealed that both tumors contained an amplified Ki-ras gene, which in one case was coamplified with c-myc. In one of the tumors, Ki-ras was found to be amplified in both the primary tumor and three different metastatic nodules. No mutations at codons 12 or 61 of Ki-ras were detected in these tumors. No additional cases of Ki-ras or c-myc amplification were detected by Southern hybridization in the tumors that were found to be amplification negative by modified in-gel renaturation assays. These results indicate that gene amplification in ovarian carcinomas is likely to involve the Ki-ras oncogene.

A senescence-like phenotype distinguishes tumor cells that undergo terminal proliferation arrest after exposure to anticancer agents.

Exposure of human tumor cell lines to different chemotherapeutic drugs, ionizing radiation, and differentiating agents induced morphological, enzymatic, and ploidy changes resembling replicative senescence of normal cells. Moderate doses of doxorubicin induced this senescence-like phenotype (SLP) in 11 of 14 tested cell lines derived from different types of human solid tumors, including all of the lines with wild-type p53 and half of p53-mutated cell lines. SLP induction seemed to be independent from mitotic cell death, the other major effect of drug treatment. Among cells that survived drug exposure, SLP markers distinguished those cells that became terminally growth-arrested within a small number of cell divisions from the cells that recovered and resumed proliferation. SLP induction in breast carcinoma cells treated with retinoids in vitro or in vivo was found to correlate with permanent growth inhibition under the conditions of minimal cytotoxicity, suggesting that this response may be particularly important for the antiproliferative effect of differentiating agents. The senescence-like program of terminal proliferation arrest may provide an important determinant of treatment outcome and a target for augmentation in cancer therapy.

Polyamine depletion in human melanoma cells leads to G1 arrest associated with induction of p21WAF1/CIP1/SDI1, changes in the expression of p21-regulated genes, and a senescence-like phenotype.

The cell cycle regulatory events that interface with polyamine requirements for cell growth have not yet been clearly identified. Here we use specific inhibitors of polyamine biosynthetic enzymes to investigate the effect of polyamine pool depletion on cell cycle regulation. Treatment of MALME-3M cells with either the ornithine decarboxylase inhibitor alpha-difluoromethylornithine or the S-adenosylmethionine decarboxylase inhibitor MDL-73811 lowered specific polyamine pools and slowed cell growth but did not induce cell cycle arrest. By contrast, treatment with the combination of inhibitors halted cell growth and caused a distinct G1 arrest. The latter was associated with marked reduction of all three polyamine pools, a strong increase in p21(WAF1/CIP1/SDI1) (p21), and hypophosphorylation of retinoblastoma protein. All effects were fully prevented by exogenous polyamines. p21 induction preceded p53 stabilization in MALME-3M cells and also occurred in a polyamine-depleted, p53-nonfunctional melanoma cell line, indicating that p21 is induced at least in part through p53-independent mechanisms. Conditional overexpression of p21 in a fibrosarcoma cell line was shown previously to inhibit the expression of multiple proliferation-associated genes and to induce the expression of genes associated with various aspects of cell senescence and organism aging. Polyamine depletion in MALME-3M cells was associated with inhibition of seven of seven tested p21-inhibited genes and with induction of 13 of 14 tested p21-induced genes. p21 expression is also known to induce a senescence-like phenotype, and phenotypic features of senescence were observed in polyamine-depleted MALME-3M cells. Cells increased in size, appeared more granular, and expressed senescence-associated beta-galactosidase. Cells released from the polyamine inhibition lost the ability to form colonies, failed to replicate their DNA, and approximately 25% became bi- or multinucleated. These events parallel the outcome of prolonged p21 induction in fibrosarcoma cells. The results of this study indicate that polyamine pool depletion achieved by specific biosynthetic enzyme inhibitors causes p21-mediated G1 cell cycle arrest followed by p21-mediated changes in gene expression, development of a senescence-like phenotype, and loss of cellular proliferative capacity.

Genetic suppressor elements: new tools for molecular oncology--thirteenth Cornelius P. Rhoads Memorial Award Lecture.

Genetic suppressor elements (GSEs) are short biologically active gene fragments that encode dominantly acting peptides or inhibitory antisense RNAs. GSEs can be isolated from a single gene or from a multigene complex by constructing a library of short random fragments of the target gene(s) in an expression vector, followed by expression selection for the desired phenotype in a suitable cellular system. GSE selection from a single gene allows one to develop efficient and specific inhibitors of the gene function and to identify functional protein domains. GSE selection from a multigene complex, such as a normalized (uniform abundance) cDNA population from mammalian cells, makes it possible to identify genes that are involved in selectable cellular phenotypes. The potential of GSE selection for uncovering novel gene functions was first demonstrated using bacteriophage lambda as a model system. GSE selection in retroviral expression vectors has been applied in mammalian cells to identify genes responsible for sensitivity to etoposide and other chemotherapeutic drugs. GSE selection is also useful for cloning and analysis of tumor suppressor genes and can be applied to identifying tumor-specific targets for future anticancer drugs. Investigators should find this experimental strategy applicable to many different areas of medical and biological research.

Relationship of the expression of the multidrug resistance gene product (P-glycoprotein) in human colon carcinoma to local tumor aggressiveness and lymph node metastasis.

P-glycoprotein mediates classic multidrug resistance by functioning as an efflux pump that excretes lipophilic chemotherapeutic drugs from cancer cells. We now report an association of P-glycoprotein in colon carcinomas with another tumor property, i.e., enhancement of local tumor aggressiveness. P-glycoprotein was detected with monoclonal antibody immunohistochemistry in 65 of 95 primary colon adenocarcinomas, which were stage B1 or greater. In all but 1 of the 95 cases, solitary invading carcinoma cells were present at the leading edge of the tumor. This subpopulation of invasive carcinoma cells expressed P-glycoprotein (P-Gp+) in 47 of the 95 surgically resected colon specimens. Cases were grouped on the basis of the presence (Group 1, 47 cases) or absence (Group 2, 48 cases) of P-Gp+ invasive carcinoma cells. There was a significantly greater incidence of vessel invasion (P less than 0.001) and lymph node metastases (P less than 0.01) in Group 1 cases. Groups 1 and 2 did not differ with respect to tumor size, depth of invasion of the bowel wall, histological grade, maximum tumor size, mitotic index, mucin production, or presence of perineural invasion (P greater than 0.1). Our findings indicate that P-Gp+ invasive colon cancer cells may have an increased potential for dissemination, suggesting that P-glycoprotein may influence cell behavior.

Identification of p53 genetic suppressor elements which confer resistance to cisplatin.

Loss of p53 function is associated with the acquisition of cisplatin resistance in the human ovarian adenocarcinoma A2780 cell line. Selection for cisplatin resistance of A2780 cells was used to isolate genetic suppressor elements (GSEs) from a retroviral library expressing random fragments of human or murine TP53 cDNA. Six GSEs were identified, encoding either dominant negative mutant peptides or antisense RNA molecules which corresponded to various regions within the TP53 gene. Both types of GSE induced cisplatin resistance when introduced individually into A2780 cells. Expression of antisense GSEs led to decreased intracellular levels of p53 protein. One sense GSE induced loss of p53-mediated activities such as DNA damage induced cell cycle arrest and apoptosis. A synthetic peptide, representing the predicted amino acid sequence of this GSE, conferred resistance to cisplatin when introduced into A2780 cells and inhibited the sequence specific DNA binding activity of p53 protein in vitro. Overall, these results directly indicate that inactivation of p53 function confers cisplatin resistance in these human ovarian tumour cells. We have identified short structural domains of p53 which are capable of independent functional interactions and highlighted the efficacy of this approach to discriminate biologically active GSEs from a random fragment library.

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.