Andrei L. Gartel, PhD
Assistant Professor
PhD, Institute of Virology, Moscow, Russia

Contact Information: agartel@uic.edu

The regulation of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1); Identification of small molecules that can target cancer progression and viral replication.

Regulation of expression of CDK inhibitor p21 (WAF1/CIP1)

The CDK inhibitor p21 (also called WAF1, CAP20, Cip1, and Sdi1) is the founding member of the Cip/Kip family of cyclin kinase inhibitors (CKIs), which also includes p27(Kip1) and p57(Kip2). These CKIs can bind and inhibit a broad range of Cyclin-Cdk complexes, and p21 overexpression leads to G1-, G2- or S-phase arrest. Transcription of the p21 gene can be activated by p53 after DNA-damage and by several p53-independent mechanisms.

We discovered a novel mechanism of p21 induction by E2F transcription factors; this mechanism may mediate p53-independent induction of p21 by the activated oncogene V12-Ras. To further elucidate the consequences of E2F1-regulated induction of p21, we developed cell lines with a tamoxifen-dependent form of E2F1. We confirmed direct interaction of E2F1 with the proximal region of the p21 promoter. Elevated E2F1 activity was sufficient to arrest a substantial subset of cells in S phase and this effect was correlated to and dependent on the induction of p21protein. Since E2F proteins control genes required for cell cycle progression and are activated by various oncogenic events, we believe that the p21-dependent arrest represents an additional mechanism that guards against unrestricted cell proliferation.

Ectopic expression of proto-oncogene c-Myc alleviates G1 cell cycle arrest. c-Myc can promote proliferation and contributes to the development of many cancers. We discovered that c-Myc can repress transcription of the p21 gene. We found that Myc represses p21 through the very short GC-rich region of its promoter just upstream of the transcription start site, although Myc does not appear to bind to the p21 promoter. This region of the p21 promoter contains multiple Sp1 binding sites, a potential Inr site and no canonical c-Myc binding sites. Our data indicate that c-Myc repression of p21 transcription is not based on interaction between the Inr-binding protein TFII-I and c-Myc, and it is independent of histone deacetylase activity. We also found that c-Myc does not need to heterodimerize with Max for repression of the p21 promoter. Coimmunoprecipitation and GST pull-down experiments demonstrated that c-Myc may form complexes with Sp1/Sp3. This suggests that Myc may repress p21 transcription not by DNA-binding, but by interactions with positive transcription factors Sp1/Sp3. This is the first evidence of interactions between Sp1/Sp3 and c-Myc and a novel mechanism of how c-Myc may repress transcription.

The anti-diabetic thiozolidinedione compound pioglitazone, a peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonist, has been found to have growth inhibitory effects in some cancer cells. The mechanism underlying this growth suppression is not well understood. In this study, we evaluated the effect of pioglitazone on p53 and p21 expression in various cancer cell lines with different p53 status. The cells with wild type p53 did not show any change in p53 levels in response to pioglitazone. Also, none of the cell lines exhibited p53-dependent or independent transcriptional induction of p21 by pioglitazone. However, PC3, an androgen-insensitive prostate cancer cell line with deletion of p53 showed an appreciable post-transcriptional induction of p21 expression after treatment with pioglitazone. These results imply that pioglitazone generally does not modulate p21 transcription in human cancer cell lines.

We identified a novel alternate mouse p21 transcript that is conserved in evolution. It differs from the classical p21 transcript in the first exon, which is located at approximately 2.8 kb upstream of transcriptional start site and is sandwiched between two p53 binding sites. This novel p21 transcript is present in most mouse tissues with highest levels of expression in spleen. In contrast to the classical p21 transcript, this new transcript is highly dependent on p53 for its basal expression, as evidenced by its absence in nearly all of p53-null mouse tissues. Expression of the novel transcript was also p53-dependent in a 10-1 mouse cell line lacking endogenous p53 and harboring temperature sensitive p53 mutant. In addition we showed that at least three different alternate p21 transcripts originate from a similar region in human cells and these transcripts also depend on p53 for their basal expression. Our data suggest that p53-dependent induction of p21 may be an additive effect conferred by individual increases in the alternate and classical p21 transcripts.

We found that CDK9 phosphorylates p53 on serine residues 33, 315 and 392. Tumor suppressor p53 is often activated in response to DNA damage or other forms of stress, leading to either cell cycle arrest or apoptosis. Stress-induced kinases phosphorylate p53 thereby enhancing its stability, leading to an increase in transactivation of its target genes. Several different protein kinases phosphorylate p53 on multiple amino acid residues. We discovered for the first time that cyclin dependent kinase 9, whose well-known substrate is RNA polymerase II, can also phosphorylate p53. Specifically, Ser33 on the N-terminus and, Ser315 and Ser392 on the C-terminus of p53 were found to be phosphorylated. The precise biological role of this phosphorylation remains to be elucidated.

Selected publications on p21

Gartel A.L. , Goufman, E., Tevosian S., Shih H. S., Yee A., and Tyner A.L. Opposing effects of different Rb binding proteins on p21 WAF1/CIP1 transcription. Oncogene, 17, 3463-70, 1998.
*Gartel A.L. and Tyner A.L. Transcriptional regulation of the p21 (WAF1/CIP1) gene . Exp. Cell Res., 246, 280-289, 1999.
*Gartel A.L., Najmabadi F., Goufman E., and Tyner A.L.* A role for E2F1 in Ras activation of p21(WAF1/CIP1) transcription. Oncogene , 19, 961-964, 2000.
* Gartel A.L. , Goufman E., Najmabadi F., Tyner A.L. Sp1 and Sp3 regulate p21(WAF1/CIP1) gene transcription in the Caco-2 colon adenocarcinoma cell line. Oncogene, 19, 5182-5188, 2000.
*Gartel A.L., Ye X., Goufman E., Shianov P., Hay N., Najmabadi F., and A. L. Tyner. 2001. Repression of the p21 WAF1/CIP1 promoter by c-Myc does not require DNA-binding and interaction with Max. Oncology Research , 12, 67, p. 293, 2001.
*Gartel A.L. , Ye X., Goufman E., Shianov P., Hay N., Najmabadi F., and Tyner A.L. Myc represses the p21 WAF1/CIP1 promoter and interacts with Sp1/Sp3. Proc Natl Acad Sci U S A. 98(8):4510-5, 2001.
*Gartel, A.L., and A. L. Tyner. The Role for the Cdk Inhibitor p21 in Apoptosis. Molecular Cancer Therapeutics , 1: 639-649, 2002.
Kandel E.S., Skeen J., Majewski N., Di Cristofano A., Pandolfi P. P., Feliciano C. S., Gartel A. L. and N. Hay. Activation of Akt/PKB abrogates G2/M cell cycle checkpoint following DNA damage. Molecular and Cellular Biology 22(22):7831-7841, 2002.
*Gartel A. L., Radhakrishnan S.K , Feliciano C., Najmabadi F., Kandel E.S., Park J.H.Y and A. L. Tyner. E2F1-dependent transactivation of p21 results in S-phase cell cycle arrest. Miami Nature Biotechnology Short Reports , 14, p. 88, 2003.
*Gartel A. L . and Shchors K. Mechanisms of c-myc-mediated transcriptional repression of growth arrest genes. Exp. Cell Res. 283:17-21, 2003.
*Gartel A. L ., Feliciano C., and A. L. Tyner. A new method for determining the status of p53 in tumor cell lines of different origin. Oncology Research , 13, 405-408, 2003.
*Tyner A. L. and A. L. Gartel*. The Role of CKIs in G1 Phase Progression. In "G1 Phase progression." Johannes Boonstra, Editor. Landes Bioscience /KluwerAcadenic/ Plenum publishers, 58-76, 2003.
Radhakrishnan S.K. Feliciano C.S., Najmabadi F., Haegebarth A., Kandel E.S., Tyner,A.L. and A. L. Gartel*. Constitutive expression of E2F-1 leads to p21-dependent cell cycle arrest in S-phase of the cell cycle. Oncogene, 23(23): 4173-6, 2004.
Datta A, Nag A, Pan W, Hay N, Gartel A. L. Colamonici O, Mori Y, Raychaudhuri P. Myc-ARF interaction inhibits the functions of Myc. J Biol Chem ., 279, 35, 36698-36707, 2004.
* Gartel A. L., Radhakrishnan S.K., Serfas M.S., Kwon Y.H., and A. L. Tyner . A novel p21 (WAF1/CIP1)  transcript is highly dependent on p53 for its basal expression in mouse tissues. Oncogene . 23(49): 8154-7, 2004.
S. K. Radhakrishnan and A. L. Gartel* . The PPAR-?amma?Agonist Pioglitazone Post Trancriptionally Induces p21 in PC3 Prostate Cancer But Not in Other Cell Lines. Cell Cycle , 4, 4, 582-4, 2005.
* Gartel A. L. and S.K. Radhakrishnan. Lost in transcription: p21 repression, mechanisms and consequences. Cancer Research , 65(10): 3980-5, 2005.
Gartel A. L. The Conflicting Roles of the Cdk Inhibitor p21 (CIP1/WAF1) in Apoptosis. Leukemia Research. 29 (11): 1237-8, 2005.
Gartel A. L . A new mode of transcriptional repression by c-Myc: methylation. Oncogene , 25:1989-90, 2006.
Radhakrishnan S.K. , and A. L. Gartel*. CDK9 phosphorylates p53 on serine residues 33, 315 and 392. Cell Cycle , 5, 5, 519-521, 2006.
Radhakrishnan S.K. , Gierut J. and A.L. Gartel*. Multiple alternate p21 transcripts are regulated by p53 in human cells. Oncogene , 25:1812-5, 2006.
Gartel A.L. Inducer and inhibitor: “antagonistic duality” of p21 in differentiation . Leukemia Research. 30(10):1215-6, 2006.
Gartel A. L. Is p21 an oncogene? Molecular Cancer Therapeutics , 5(6), 1385-1386, 2006.
Gartel A. L. p21 may be a tumor suppressor after all. Cancer Biology &Therapy , 6(8): 1171-252, 2007.

Identification of novel anticancer drugs

Conventional treatments for human cancer are often fraught with side effects and limited efficiency, and repeatedly fail to achieve long term patients survival. Therefore, further identification of novel therapeutic strategies for the treatment of cancer is extremely important. Using a high-throughput cell-based assay, we identified a nucleoside analog ARC (4-amino-6-hydrazino-7-beta-D-ribofuranosyl-7H-Pyrrolo[2,3-d]-pyrimidine-5-carboxamide), which has properties of a general transcriptional inhibitor. Specifically, ARC inhibits phosphorylation of RNA polymerase II by PTEF-b (positive transcription elongation factor b) leading to a block in transcriptional elongation. ARC was able to potently repress p53 targets p21 and hdm2 (human homolog of mdm2) protein levels, but dramatically increased p53 levels similar to other transcriptional inhibitors, including flavopiridol. This increase in p53 corresponded to the downregulation of short-lived protein hdm2, which is a well-established negative regulator of p53. Remarkably, ARC induced potent apoptosis in human tumor and transformed, but not in normal cells and possessed strong anti-angiogenic activity in vitro. Although ARC promoted accumulation of p53, ARC-induced apoptosis in tumor cells was p53-independent, suggesting that it may be useful for treatment of tumors with functionally inactive p53. Furthermore, cell death induced by ARC had a strong correlation with downregulation of anti-apoptotic proteins survivin and Mcl-1, which are often overexpressed in human tumors. In addition, we found that neuroblastoma cell lines are susceptible to ARC in nanomolar concentrations and N-myc protein levels were reduced in response to ARC treatment. This is especially significant given the fact that one-third of neuroblastoma tumors shows amplification of N-myc, which has a strong correlation with chemotherapy resistance Taken together, our data suggest that ARC may be an attractive candidate for anti-cancer drug development. Additionally, we found that ARC has the ability to inhibit replication of HCV and HIV.

We also used another approach to identify potential anticancer drugs. It has been shown that FoxM1, a transcription factor of the Forkhead family is one of the most upregulated genes in human solid tumors and is implicated in tumor invasion, angiogenesis and metastasis. Since FoxM is activated in the majority of cancers, but not in normal cells, we hypothesized that it could be an attractive target for anticancer therapy by small molecules. We designed a novel screening system for the identification of compounds that will inhibit transcriptional activation of genes by FoxM1 . We created a cell line that stably expresses doxycycline-inducible FoxM1-GFP fusion protein, firefly luciferase under the control of multiple FoxM response elements, and control renilla luciferase under the CMV promoter. We screened libraries of small molecules from NCI for inhibitors of FoxM1 transactivation utilizing this cell line as a read-out system. As a result of the screening we identified a thiazole antibiotic , Siomycin A as an inhibitor of FoxM1 transcriptional activity. Thiazole antibiotics exert their antibacterial effects by inhibiting bacterial translation via interaction with the 23S ribosomal RNA, but they do not inhibit eukaryotic translation. To examine the anticancer potential of the thiazole antibiotics we studied their effect on human cancer cell lines of different origin. Activation of apoptosis by thiazole antibiotics correlated with suppression of FoxM1 protein, suggesting that thiopeptides may induce apoptosis to a certain extent through the inhibition of FoxM1. Overall, our data suggest that potentially FoxM inhibitors/thiazole antibiotics could be useful for cancer treatment, but additional experiments are needed to determine if FoxM inhibitors are feasible alternative for cancer patients treatment. As proof of principle, we decided to test ARC and the thiazole antibiotics against metastatic melanoma cells in vitro. Our data suggest that the melanoma cells are much more sensitive to ARC and to the thiazole antibiotics than to DTIC, a well-known anti-melanoma drug.

Selected Publications on anticancer drugs and RNA interference:

Radhakrishnan S.K., Layden TJ, and A. L. Gartel* . RNA interference as a new strategy against viral hepatitis . Virology, 323, 173-181, 2004.
Radhakrishnan S.K. , and A.L. Gartel*. A novel transcriptional inhibitor induces apoptosis in tumor cells and exhibits anti-angiogenic activity (Featured in the section Cancer Research Highlights: Selected articles from this issue ) Cancer Research , 66, 6, 3264-70, 2006.
* Gartel A. L. and Kandel E.S. RNA interference in cancer. Biomolecular Engineering 23, 1, 17-34, 2006.
Radhakrishnan S.K. , Bhat, U.G., Hughes D. E., I-Ching Wang , Costa R. H. and A. L. Gartel*. Identification of a chemical inhibitor of the oncogenic transcription factor Forkhead Box M1. (Featured in the section Cancer Research Highlights: Selected articles from this issue ) Cancer Research, 66, 19, 9731-5, 2006.
Nekhai S., Bhat UG, Ammosova T, Radhakrishnan SK., Jerebtsova M., Niu X, Foster A., Layden TJ, and A. L. Gartel* Novel transcriptional inhibitor ARC antagonizes HIV-1 and HCV, Oncogene , 2007, 26(26):3899-903.
Radhakrishnan SK, Halasi M, Bhat UG, Kurmasheva RT, Houghton PJ, and A. L. Gartel*. Proapoptotic compound ARC targets Akt and N-myc in neuroblastoma cells. Oncogene , 27(5):694-9, 2008.
Radhakrishnan SK, Bhat UG, Halasi M and A. L. Gartel*. P-TEFb inhibitors interfere with activation of p53 by DNA-damaging agents. Oncogene , 27(9):1306-9, 2008.
Bhat UG, and A. L. Gartel* Differential sensitivity of human colon cancer cell lines to the nucleoside analogs ARC and DRB . International Journal of Cancer , 122(6):1426-1429, 2008.
Gartel A.L . and Kandel E.S. miRNAs: little known mediators of oncogenesis. Seminars in Cancer Biology , 18, 103-110, 2008.
Radhakrishnan SK and Gartel A.L . FoxM1: The Achilles' heel of Cancer? Nat Rev Cancer , 8 (3), 2008 http://www.nature.com/nrc/journal/v8/n3/full/nrc2223-c1.html .
Bhat UG, Zipfel PA, Tyler DC and A.L. Gartel. Novel anticancer compounds induce apoptosis in melanoma cells. Cell Cycle , June 15, Volume: 7/ Issue: 12, 2008.
Gartel A.L. Forkhead Box M1 “Encyclopedia of Cancer” (Ed. Manfred Schwab), Springer, Berlin-Heidelberg-New York, 2008.
Gartel A.L. Novel anticancer compounds induce apoptosis in human tumor cells, Chinese Journal of Cancer, volume 27/ issue 7, 2008.
Gartel A.L . Transcriptional inhibitors, p53 and apoptosis. BBA-Cancer Reviews , (in press) 2008.
Gartel A.L . FoxM1 inhibitors as potential anticancer drugs. Expert Opinion on Therapeutic Targets , 6, 12, 2008.