CDK8 and CDK19 are kinase components of the CDK module that comprises CDK8 or CDK19 together with... more CDK8 and CDK19 are kinase components of the CDK module that comprises CDK8 or CDK19 together with their binding partner cyclin C (CCNC) and two other proteins; the CDK module is associated with transcriptional Mediator complex. CDK8/19 Mediator kinases potentiate transcription induced by different signals, regulate protein levels of Mediator complex components and protect CCNC from proteolytic degradation; the latter activity is exerted in a kinase-independent manner. CDK8/19 kinase inhibitors have entered clinical trials for solid tumors and leukemias. To extend the effects of CDK8/19 inhibition and to suppress their kinase-independent activities, we have developed three series of PROteolysis TArgeting Chimeras (PROTACs) based on different selective inhibitors of CDK8/19 kinases, connected via different linkers to a moiety binding cereblon (CRBN) E3 ligase. The most potent PROTACs degraded both CDK8 and CDK19 in different cell types with DC50s of 10-20 nM. RNA-Seq analysis of 293 cells treated with a CDK8/19 kinase inhibitor or PROTAC showed that the bulk of the PROTAC’s transcriptomic effects matched the effects of the kinase inhibitor. In contrast to the kinase inhibitors, CDK8/19-degrading PROTACs also induced CCNC degradation. Cancer Dependency Map (DepMap) analysis revealed that multiple myelomas (MM), where CRBN is an established therapeutic target, show greater dependency on CCNC than most of the tumor cell lines. We have tested two strongly CCNC-dependent MM cell lines and one CCNC-independent MM line for sensitivity to different CDK8/19 kinase inhibitors and PROTACs. Kinase inhibitors showed low to moderate anti-proliferative activity in all three MM lines, whereas CDK8/19 PROTACs were an order of magnitude more potent than the kinase inhibitors in the two CCNC-dependent MM lines (IC50s of 20-30 nM) but not in the CCNC-independent line. The effects of CDK8/19-degrading PROTACs were stronger than those of the CRBN-targeting drug pomalidomide (approved for MM), a CRBN-binding PROTAC acting on androgen receptor or a CDK8 degradation-inactive PROTAC analog containing CDK8/19- and CRBN-binding moieties. These results suggest the potential of CDK8/CDK19-degrading PROTACs for the treatment of MM. Citation Format: Li Zhang, Jing Li, Charles E. Dowling, Eugenia V. Broude, Igor B. Roninson, Campbell McInnes, Mengqian Chen. PROTAC degraders of CDK8/CDK19 Mediator kinases potently suppress multiple myeloma proliferation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5018.
Proceedings of the National Academy of Sciences of the United States of America, Aug 1, 2022
Breast cancers (BrCas) that overexpress oncogenic tyrosine kinase receptor HER2 are treated with ... more Breast cancers (BrCas) that overexpress oncogenic tyrosine kinase receptor HER2 are treated with HER2-targeting antibodies (such as trastuzumab) or small-molecule kinase inhibitors (such as lapatinib). However, most patients with metastatic HER2+BrCa have intrinsic resistance and nearly all eventually become resistant to HER2-targeting therapy. Resistance to HER2-targeting drugs frequently involves transcriptional reprogramming associated with constitutive activation of different signaling pathways. We have investigated the role of CDK8/19 Mediator kinase, a regulator of transcriptional reprogramming, in the response of HER2+BrCa to HER2-targeting drugs. CDK8 was in the top 1% of all genes ranked by correlation with shorter relapse-free survival among treated HER2+BrCa patients. Selective CDK8/19 inhibitors (senexin B and SNX631) showed synergistic interactions with lapatinib and trastuzumab in a panel of HER2+BrCa cell lines, overcoming and preventing resistance to HER2-targeting drugs. The synergistic effects were mediated in part through the PI3K/AKT/mTOR pathway and reduced by PI3K inhibition. Combination of HER2- and CDK8/19-targeting agents inhibited STAT1 and STAT3 phosphorylation at S727 and up-regulated tumor suppressor BTG2. The growth of xenograft tumors formed by lapatinib-sensitive or -resistant HER2+breast cancer cells was partially inhibited by SNX631 alone and strongly suppressed by the combination of SNX631 and lapatinib, overcoming lapatinib resistance. These effects were associated with decreased tumor cell proliferation and altered recruitment of stromal components to the xenograft tumors. These results suggest potential clinical benefit of combining HER2- and CDK8/19-targeting drugs in the treatment of metastatic HER2+BrCa.
The Senexins are potent and selective quinazoline inhibitors of CDK8/19 Mediator kinases. To impr... more The Senexins are potent and selective quinazoline inhibitors of CDK8/19 Mediator kinases. To improve their potency and metabolic stability, quinoline-based derivatives were designed through a structure-guided strategy based on the simulated drug-target docking model of Senexin A and B. A library of quinoline-Senexin derivatives was synthesized to explore the structure-activity relationship. An optimized compound 20a (Senexin C) exhibits potent CDK8/19 inhibitory activity with a high selectivity. Senexin C is more metabolically stable and provides a more sustained inhibition of CDK8/19-dependent cellular gene expression when compared with the prototype inhibitor Senexin B. In vivo pharmacokinetic (PK) and pharmacodynamic (PD) evaluation using a novel tumor-based PD assay showed good oral bioavailability of Senexin C with a strong tumor-enrichment PK profile and tumor-PD marker responses. Senexin C inhibits MV4-11 leukemia growth in a systemic in vivo model with good tolerability.
SY21-3 When damaged by chemotherapy or radiation, a tumor cell may (i) become arrested at a cell ... more SY21-3 When damaged by chemotherapy or radiation, a tumor cell may (i) become arrested at a cell cycle checkpoint, repair the damage and resume proliferation, (ii) proceed directly into apoptosis or some other form of programmed cell death, (iii) undergo permanent cell cycle arrest through the program of senescence, or (iv) undergo one of many forms of abnormal mitosis - mitotic catastrophe. Direct (pre-mitotic) induction of apoptosis after exposure to conventional chemotherapeutics or radiation appears to be the least common outcome in the majority of cell lines derived from human solid tumors. Senescence is induced very efficiently by DNA-interactive compounds but less efficiently by anti-microtubular drugs. In contrast, mitotic catastrophe is a general and common response to all the major classes of anticancer agents. The outcome of treatment depends not only on whether tumor cells undergo mitotic catastrophe or senescence but also on the specific pathways and phenotypes of these responses. In particular, mitotic catastrophe may occur through different pathways depending both on its inducing agent and on the status of different cell cycle checkpoints. As a result, different pathways of mitotic catastrophe may result in apoptosis, necrosis, senescence or even recovery of cells with altered DNA content. Phenotypic characterization is even more important in the case of senescence, since senescent tumor cells, although not dividing, are very long-lived and secrete various proteins that affect the tumor environment. Some of these proteins inhibit cell growth or carcinogenesis but others have the opposite effect on neighboring cells, promoting cell division, survival or invasive growth. Several proteins overexpressed by senescent cells have also been implicated in various age-related diseases. Senescent cells differ in their relative levels of expression of different bioactive proteins and the corresponding paracrine activities. Expression of many senescence-associated proteins with disease-promoting activities is mediated by CDK inhibitor proteins p21(Waf1/Cip1/Sdi1) and p16(Ink4a). The effects of CDK inhibitors on gene expression are exerted at the level of transcription, with transcription factor NFκB playing an important role in the induction of disease-associated genes. The emerging knowledge about the mechanisms of mitotic catastrophe and transcriptional regulation in senescent cells suggests new approaches to improving the therapeutic index in cancer treatment.
We discuss our recent findings in three related areas of the gene amplification field. 1) We have... more We discuss our recent findings in three related areas of the gene amplification field. 1) We have found that tumor-promoting phorbol esters, nonphorbol tumor promoters, and most significantly, mitogenic hormones, such as insulin, vasopressin, and epidermal growth factor (EGF), greatly increase the incidence of methotrexate (MTX) resistance in 3T6 cells under condition of MTX selection. Most of these MTX-resistant cells bear amplified dihydrofolate reductase (DHFR) genes. 2) We have discovered that when mouse cells bearing unstably amplified DHFR genes are grown in the presence of nonlethal concentrations of hydroxyurea (HU), the rate of loss of the DHFR genes from these cells is greatly increased. 3) We have developed a new method for detection and mapping of homologous, repeated and amplified DNA sequences, and have used this method to detect and clone amplified DNA fragments in mammalian cells resistant simultaneously to a number of different drugs.
We have investigated the presence or absence of methylation of cytosine (5-MeCpG) at specific sit... more We have investigated the presence or absence of methylation of cytosine (5-MeCpG) at specific sites of the chick alpha-globin gene cluster in DNA from embryonic and adult erythroid cells, as well as from brain and sperm cells. We find that these sites are totally methylated in sperm DNA and, with some exceptions, in brain DNA. In erythroid cells no methylation or undermethylation is found in certain sites that are in or near those globin genes that are expressed in embryonic or adult cells, respectively, as predicted by the model. Other sites, away from these genes, do not necessarily follow the prediction. We have also detected a cluster of 5-MeCpC methylation sites in all tissues which is 5' to the alpha-globin cluster.
Perivascular delivery of therapeutic agents against established aetiologies for occlusive vascula... more Perivascular delivery of therapeutic agents against established aetiologies for occlusive vascular remodelling has great therapeutic potential for vein graft failure. However, none of the perivascular drug delivery systems tested experimentally have been translated into clinical practice. In this study, we established a novel strategy to locally and sustainably deliver the cyclin-dependent kinase 8/19 inhibitor Senexin A (SenA), an emerging drug candidate to treat occlusive vascular disease, using graphene oxide-hybridised hyaluronic acid-based hydrogels. We demonstrated an approach to accommodate SenA in hyaluronic acid-based hydrogels through utilising graphene oxide nanosheets allowing for non-covalent interaction with SenA. The resulting hydrogels produced sustained delivery of SenA over 21 days with tunable release kinetics. In vitro assays also demonstrated that the hydrogels were biocompatible. This novel graphene oxide-incorporated hyaluronic acid hydrogel offers an optimistic outlook as a perivascular drug delivery system for treating occlusive vascular diseases, such as vein graft failure.
Induction of p21 (WAF1/CIP1/SDI1), a physiological mediator of cell cycle arrest, inhibits multip... more Induction of p21 (WAF1/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 IIalpha (TOPO IIalpha) p21 expression from an inducible promoter in human HT1080 cells rapidly decreases cellular levels of PLK1 and TOPO IIalpha 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 IIalpha 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 con-sequence of cell cycle arrest.
P-glycoprotein, encoded by the MDR1 (multidrug resistance) gene, is a transmembrane efflux pump f... more P-glycoprotein, encoded by the MDR1 (multidrug resistance) gene, is a transmembrane efflux pump for various lipophilic compounds. MDR1 is expressed in several types of normal human tissues and in a variety of tumors, where its expression has been correlated with resistance to chemotherapy. Some P-glycoprotein-overexpressing multidrug-resistant cell lines contain elevated amounts of protein kinase C (PKC). PKC activation was shown to increase the level of drug resistance in several cell lines, but the functional association of PKC with P-glycoprotein-mediated multidrug resistance remains unclear. We have studied the effects of lymphocyte-activating agents on P-glycoprotein activity in normal human lymphocytes, and found that 12-O-tetradecanoylphorbol-13-acetate (TPA), an efficient agonist of PKC, increased the activity as well as the levels of P-glycoprotein in these cells. TPA also increased P-glycoprotein expression in several cell lines derived from different types of leukemias and solid tumors. The increase in MDR1 gene expression was observed at both the protein and RNA levels. Induction of MDR1 mRNA was apparent as early as two hours after the addition of TPA. Diacylglycerol (DAG), a physiological stimulant of PKC, also increased the expression of MDR1 mRNA and P-glycoprotein. The induction of MDR1 expression by TPA and DAG was suppressed by staurosporine, a protein kinase inhibitor. The results suggest that MDR1 gene expression in different cell types is regulated by a PKC-mediated pathway. This finding has implications for the emergence of multidrug resistance in vitro and in vivo.
P-glycoprotein mediates classic multidrug resistance by functioning as an efflux pump that excret... more 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.
CDK8 and CDK19 are kinase components of the CDK module that comprises CDK8 or CDK19 together with... more CDK8 and CDK19 are kinase components of the CDK module that comprises CDK8 or CDK19 together with their binding partner cyclin C (CCNC) and two other proteins; the CDK module is associated with transcriptional Mediator complex. CDK8/19 Mediator kinases potentiate transcription induced by different signals, regulate protein levels of Mediator complex components and protect CCNC from proteolytic degradation; the latter activity is exerted in a kinase-independent manner. CDK8/19 kinase inhibitors have entered clinical trials for solid tumors and leukemias. To extend the effects of CDK8/19 inhibition and to suppress their kinase-independent activities, we have developed three series of PROteolysis TArgeting Chimeras (PROTACs) based on different selective inhibitors of CDK8/19 kinases, connected via different linkers to a moiety binding cereblon (CRBN) E3 ligase. The most potent PROTACs degraded both CDK8 and CDK19 in different cell types with DC50s of 10-20 nM. RNA-Seq analysis of 293 cells treated with a CDK8/19 kinase inhibitor or PROTAC showed that the bulk of the PROTAC’s transcriptomic effects matched the effects of the kinase inhibitor. In contrast to the kinase inhibitors, CDK8/19-degrading PROTACs also induced CCNC degradation. Cancer Dependency Map (DepMap) analysis revealed that multiple myelomas (MM), where CRBN is an established therapeutic target, show greater dependency on CCNC than most of the tumor cell lines. We have tested two strongly CCNC-dependent MM cell lines and one CCNC-independent MM line for sensitivity to different CDK8/19 kinase inhibitors and PROTACs. Kinase inhibitors showed low to moderate anti-proliferative activity in all three MM lines, whereas CDK8/19 PROTACs were an order of magnitude more potent than the kinase inhibitors in the two CCNC-dependent MM lines (IC50s of 20-30 nM) but not in the CCNC-independent line. The effects of CDK8/19-degrading PROTACs were stronger than those of the CRBN-targeting drug pomalidomide (approved for MM), a CRBN-binding PROTAC acting on androgen receptor or a CDK8 degradation-inactive PROTAC analog containing CDK8/19- and CRBN-binding moieties. These results suggest the potential of CDK8/CDK19-degrading PROTACs for the treatment of MM. Citation Format: Li Zhang, Jing Li, Charles E. Dowling, Eugenia V. Broude, Igor B. Roninson, Campbell McInnes, Mengqian Chen. PROTAC degraders of CDK8/CDK19 Mediator kinases potently suppress multiple myeloma proliferation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5018.
Proceedings of the National Academy of Sciences of the United States of America, Aug 1, 2022
Breast cancers (BrCas) that overexpress oncogenic tyrosine kinase receptor HER2 are treated with ... more Breast cancers (BrCas) that overexpress oncogenic tyrosine kinase receptor HER2 are treated with HER2-targeting antibodies (such as trastuzumab) or small-molecule kinase inhibitors (such as lapatinib). However, most patients with metastatic HER2+BrCa have intrinsic resistance and nearly all eventually become resistant to HER2-targeting therapy. Resistance to HER2-targeting drugs frequently involves transcriptional reprogramming associated with constitutive activation of different signaling pathways. We have investigated the role of CDK8/19 Mediator kinase, a regulator of transcriptional reprogramming, in the response of HER2+BrCa to HER2-targeting drugs. CDK8 was in the top 1% of all genes ranked by correlation with shorter relapse-free survival among treated HER2+BrCa patients. Selective CDK8/19 inhibitors (senexin B and SNX631) showed synergistic interactions with lapatinib and trastuzumab in a panel of HER2+BrCa cell lines, overcoming and preventing resistance to HER2-targeting drugs. The synergistic effects were mediated in part through the PI3K/AKT/mTOR pathway and reduced by PI3K inhibition. Combination of HER2- and CDK8/19-targeting agents inhibited STAT1 and STAT3 phosphorylation at S727 and up-regulated tumor suppressor BTG2. The growth of xenograft tumors formed by lapatinib-sensitive or -resistant HER2+breast cancer cells was partially inhibited by SNX631 alone and strongly suppressed by the combination of SNX631 and lapatinib, overcoming lapatinib resistance. These effects were associated with decreased tumor cell proliferation and altered recruitment of stromal components to the xenograft tumors. These results suggest potential clinical benefit of combining HER2- and CDK8/19-targeting drugs in the treatment of metastatic HER2+BrCa.
The Senexins are potent and selective quinazoline inhibitors of CDK8/19 Mediator kinases. To impr... more The Senexins are potent and selective quinazoline inhibitors of CDK8/19 Mediator kinases. To improve their potency and metabolic stability, quinoline-based derivatives were designed through a structure-guided strategy based on the simulated drug-target docking model of Senexin A and B. A library of quinoline-Senexin derivatives was synthesized to explore the structure-activity relationship. An optimized compound 20a (Senexin C) exhibits potent CDK8/19 inhibitory activity with a high selectivity. Senexin C is more metabolically stable and provides a more sustained inhibition of CDK8/19-dependent cellular gene expression when compared with the prototype inhibitor Senexin B. In vivo pharmacokinetic (PK) and pharmacodynamic (PD) evaluation using a novel tumor-based PD assay showed good oral bioavailability of Senexin C with a strong tumor-enrichment PK profile and tumor-PD marker responses. Senexin C inhibits MV4-11 leukemia growth in a systemic in vivo model with good tolerability.
SY21-3 When damaged by chemotherapy or radiation, a tumor cell may (i) become arrested at a cell ... more SY21-3 When damaged by chemotherapy or radiation, a tumor cell may (i) become arrested at a cell cycle checkpoint, repair the damage and resume proliferation, (ii) proceed directly into apoptosis or some other form of programmed cell death, (iii) undergo permanent cell cycle arrest through the program of senescence, or (iv) undergo one of many forms of abnormal mitosis - mitotic catastrophe. Direct (pre-mitotic) induction of apoptosis after exposure to conventional chemotherapeutics or radiation appears to be the least common outcome in the majority of cell lines derived from human solid tumors. Senescence is induced very efficiently by DNA-interactive compounds but less efficiently by anti-microtubular drugs. In contrast, mitotic catastrophe is a general and common response to all the major classes of anticancer agents. The outcome of treatment depends not only on whether tumor cells undergo mitotic catastrophe or senescence but also on the specific pathways and phenotypes of these responses. In particular, mitotic catastrophe may occur through different pathways depending both on its inducing agent and on the status of different cell cycle checkpoints. As a result, different pathways of mitotic catastrophe may result in apoptosis, necrosis, senescence or even recovery of cells with altered DNA content. Phenotypic characterization is even more important in the case of senescence, since senescent tumor cells, although not dividing, are very long-lived and secrete various proteins that affect the tumor environment. Some of these proteins inhibit cell growth or carcinogenesis but others have the opposite effect on neighboring cells, promoting cell division, survival or invasive growth. Several proteins overexpressed by senescent cells have also been implicated in various age-related diseases. Senescent cells differ in their relative levels of expression of different bioactive proteins and the corresponding paracrine activities. Expression of many senescence-associated proteins with disease-promoting activities is mediated by CDK inhibitor proteins p21(Waf1/Cip1/Sdi1) and p16(Ink4a). The effects of CDK inhibitors on gene expression are exerted at the level of transcription, with transcription factor NFκB playing an important role in the induction of disease-associated genes. The emerging knowledge about the mechanisms of mitotic catastrophe and transcriptional regulation in senescent cells suggests new approaches to improving the therapeutic index in cancer treatment.
We discuss our recent findings in three related areas of the gene amplification field. 1) We have... more We discuss our recent findings in three related areas of the gene amplification field. 1) We have found that tumor-promoting phorbol esters, nonphorbol tumor promoters, and most significantly, mitogenic hormones, such as insulin, vasopressin, and epidermal growth factor (EGF), greatly increase the incidence of methotrexate (MTX) resistance in 3T6 cells under condition of MTX selection. Most of these MTX-resistant cells bear amplified dihydrofolate reductase (DHFR) genes. 2) We have discovered that when mouse cells bearing unstably amplified DHFR genes are grown in the presence of nonlethal concentrations of hydroxyurea (HU), the rate of loss of the DHFR genes from these cells is greatly increased. 3) We have developed a new method for detection and mapping of homologous, repeated and amplified DNA sequences, and have used this method to detect and clone amplified DNA fragments in mammalian cells resistant simultaneously to a number of different drugs.
We have investigated the presence or absence of methylation of cytosine (5-MeCpG) at specific sit... more We have investigated the presence or absence of methylation of cytosine (5-MeCpG) at specific sites of the chick alpha-globin gene cluster in DNA from embryonic and adult erythroid cells, as well as from brain and sperm cells. We find that these sites are totally methylated in sperm DNA and, with some exceptions, in brain DNA. In erythroid cells no methylation or undermethylation is found in certain sites that are in or near those globin genes that are expressed in embryonic or adult cells, respectively, as predicted by the model. Other sites, away from these genes, do not necessarily follow the prediction. We have also detected a cluster of 5-MeCpC methylation sites in all tissues which is 5' to the alpha-globin cluster.
Perivascular delivery of therapeutic agents against established aetiologies for occlusive vascula... more Perivascular delivery of therapeutic agents against established aetiologies for occlusive vascular remodelling has great therapeutic potential for vein graft failure. However, none of the perivascular drug delivery systems tested experimentally have been translated into clinical practice. In this study, we established a novel strategy to locally and sustainably deliver the cyclin-dependent kinase 8/19 inhibitor Senexin A (SenA), an emerging drug candidate to treat occlusive vascular disease, using graphene oxide-hybridised hyaluronic acid-based hydrogels. We demonstrated an approach to accommodate SenA in hyaluronic acid-based hydrogels through utilising graphene oxide nanosheets allowing for non-covalent interaction with SenA. The resulting hydrogels produced sustained delivery of SenA over 21 days with tunable release kinetics. In vitro assays also demonstrated that the hydrogels were biocompatible. This novel graphene oxide-incorporated hyaluronic acid hydrogel offers an optimistic outlook as a perivascular drug delivery system for treating occlusive vascular diseases, such as vein graft failure.
Induction of p21 (WAF1/CIP1/SDI1), a physiological mediator of cell cycle arrest, inhibits multip... more Induction of p21 (WAF1/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 IIalpha (TOPO IIalpha) p21 expression from an inducible promoter in human HT1080 cells rapidly decreases cellular levels of PLK1 and TOPO IIalpha 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 IIalpha 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 con-sequence of cell cycle arrest.
P-glycoprotein, encoded by the MDR1 (multidrug resistance) gene, is a transmembrane efflux pump f... more P-glycoprotein, encoded by the MDR1 (multidrug resistance) gene, is a transmembrane efflux pump for various lipophilic compounds. MDR1 is expressed in several types of normal human tissues and in a variety of tumors, where its expression has been correlated with resistance to chemotherapy. Some P-glycoprotein-overexpressing multidrug-resistant cell lines contain elevated amounts of protein kinase C (PKC). PKC activation was shown to increase the level of drug resistance in several cell lines, but the functional association of PKC with P-glycoprotein-mediated multidrug resistance remains unclear. We have studied the effects of lymphocyte-activating agents on P-glycoprotein activity in normal human lymphocytes, and found that 12-O-tetradecanoylphorbol-13-acetate (TPA), an efficient agonist of PKC, increased the activity as well as the levels of P-glycoprotein in these cells. TPA also increased P-glycoprotein expression in several cell lines derived from different types of leukemias and solid tumors. The increase in MDR1 gene expression was observed at both the protein and RNA levels. Induction of MDR1 mRNA was apparent as early as two hours after the addition of TPA. Diacylglycerol (DAG), a physiological stimulant of PKC, also increased the expression of MDR1 mRNA and P-glycoprotein. The induction of MDR1 expression by TPA and DAG was suppressed by staurosporine, a protein kinase inhibitor. The results suggest that MDR1 gene expression in different cell types is regulated by a PKC-mediated pathway. This finding has implications for the emergence of multidrug resistance in vitro and in vivo.
P-glycoprotein mediates classic multidrug resistance by functioning as an efflux pump that excret... more 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.
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