Pankaj Kumar’s research while affiliated with University of Pennsylvania and other places

EBNA3C, one of the Epstein-Barr virus (EBV)-encoded latent antigens, is essential for primary B-cell transformation. Cyclin D1, a key regulator of G1 to S phase progression, is tightly associated and aberrantly expressed in numerous human cancers. Previously, EBNA3C was shown to bind to Cyclin D1 in vitro along with Cyclin A and Cyclin E. In the present study, we provide evidence which demonstrates that EBNA3C forms a complex with Cyclin D1 in human cells. Detailed mapping experiments show that a small N-terminal region which lies between amino acids 130-160 of EBNA3C binds to two different sites of Cyclin D1- the N-terminal pRb binding domain (residues 1-50), and C-terminal domain (residues 171-240), known to regulate Cyclin D1 stability. Cyclin D1 is short-lived and ubiquitin-mediated proteasomal degradation has been targeted as a means of therapeutic intervention. Here, we show that EBNA3C stabilizes Cyclin D1 through inhibition of its poly-ubiquitination, and also increases its nuclear localization by blocking GSK3β activity. We further show that EBNA3C enhances the kinase activity of Cyclin D1/CDK6 which enables subsequent ubiquitination and degradation of pRb. EBNA3C together with Cyclin D1-CDK6 complex also efficiently nullifies the inhibitory effect of pRb on cell growth. Moreover, an sh-RNA based strategy for knock-down of both cyclin D1 and EBNA3C genes in EBV transformed lymphoblastoid cell lines (LCLs) shows a significant reduction in cell-growth. Based on these results, we propose that EBNA3C can stabilize as well as enhance the functional activity of Cyclin D1 thereby facilitating the G1-S transition in EBV transformed lymphoblastoid cell lines.

Nm23 was first cloned from a murine melanoma cell line wherein its expression correlated inversely with metastatic potential (Steeg et al. 1988). While it is abundantly expressed in aggressive neuroblastoma, the NB alleles typically encode the S120G substitution, which is likely to be a hypomorphic variant with reduced function (Chang et al. 1994, 1996). Mutations in other members of Nm23 family proteins, although not complete loss of function, are thought to be hypomorphic as well. Furthermore, in colorectal cancers, the allelic deletions of Nm23-H1 gene have been associated with a more aggressive behavior of cancers in some studies (Campo et al. 1994) and loss of heterozygocity of Nm23-H1 gene contributes to the metastasis potential of hepatocellular carcinoma (Ye et al. 1998). The molecular mechanisms underlying the biological activity of Nm23 are delineated below.

Nm23-H1 is a well-known tumor metastasis suppressor, which functions as a nucleoside-diphosphate kinase converting nucleoside diphosphates to nucleoside triphosphates with an expense of ATP. It regulates a variety of cellular activities, including proliferation, development, migration and differentiation known to be modulated by a series of complex signaling pathway. Few studies have addressed the mechanistic action of Nm23-H1 in the context of these cellular processes. To determine the downstream pathways modulated by Nm23-H1, we expressed Nm23-H1 in a Burkitt lymphoma derived B-cell line BJAB and performed pathway specific microarray analysis. The genes with significant changes in expression patterns were clustered in groups which are responsible for regulating cell cycle, p53 activities and apoptosis. We found a general reduction of cell cycle regulatory proteins including cyclins and cyclin dependent kinase inhibitors (anti proliferation), and upregulation of apoptotic genes which included caspase 3, 9 and Bcl-x. Nm23-H1 was also found to upregulate p53 and downregulate p21 expression. A number of these genes were validated by real time PCR and results from promoter assays indicated that Nm23-H1 expression downregulated cyclin D1 in a dose responsive manner. Further, we show that Nm23-H1 forms a complex with the cellular transcription factor AP1 to modulate cyclin D1 expression levels. BJAB cells expressing Nm23-H1 showed reduced proliferation rate and were susceptible to increased apoptosis which may in part be due to a direct interaction between Nm23-H1 and p53. These results suggest that Nm23-H1 may have a role in the regulation of cell cycle and apoptosis in human B-cells.

(A) Loss of PIASy reduces the stability of VHL-SUMO1ΔC4 in hypoxia. 786-O stable cells with PIASy knockdown or luciferase control were individually transfected with VHL-SUMO1ΔC4. At 24 hr posttransfection, cells were equally divided and treated with 1% O2 for 0, 6, 12 or 24 hours. The levels of VHL-SUMO1ΔC4 were detected by immunoblotting with anti-FLAG. (B) Loss of PIASy but not Ubc9 attenuates the transcriptional activity of HIF-responsive reporter. HEK293 cells were transiently transfected with pSIREN expressing small hairpin against PIASy or Ubc9 in the presence of wild type (wHRE) or mutation (mHRE) HIF-responsive element-luciferase reporter. Empty vector was used as control. Data are presented as means±SD of three independent experiments. (2.57 MB TIF)

(A) Loss of PIASy enhances the effect of VHL positive but not negative cells on cell cycle under serum starve stress. Cells were plated and incubated for 18 hrs in either 1% oxygen or 0.1% serum, and directly analyzed by flow cytometry after staining with propidium iodide. The relative ratios of G0 phase in each group were presented by compared to luciferase knockdown (shLuc) control under normal condition. a, HeLa; b, 293; c, 786-O; d, RCC4. (B) Loss of PIASy suppresses the cell mobility in normoxia. The cell mobility of HeLa cells with PIASy stable knockdown or luciferase control in normoxia were determined by wound migration assay as described previously. (2.34 MB TIF)

The hypoxic microenvironment contributes to embryonic development and tumor progression through stabilization of the potent transcriptional factor HIFalpha. In normoxia, the tumor suppressor protein VHL acts as an E3 ubiquitin ligase to target HIFalpha for proteolytic destruction. Increasing evidence shows that VHL is a multifunctional adaptor involved in inhibition of HIFalpha-dependent and independent cellular processes. However, the molecular effect of hypoxic stress on VHL functions remains elusive. Here we report that PIASy, a SUMO E3 ligase upregulated in hypoxia, interacts with VHL and induces VHL SUMOylation on lysine residue 171. Moreover, PIASy-mediated SUMO1 modification induces VHL oligomerization and abrogates its inhibitory function on tumor cell growth, migration and clonogenicity. Knockdown of PIASy by small interfering RNA leads to reduction of VHL oligomerization and increases HIF1alpha degradation. These findings reveal a unique molecular strategy for inactivation of VHL under hypoxic stress.

The viruses associated with human cancer include Epstein Barr virus (EBV), human papilloma virus (HPV), human T-cell lymphotrophic virus (HTVL-1), Kaposi sarcoma virus (KSHV), hepatitis C virus (HCV), and hepatitis B virus (HBV). Our understanding of how these viruses operate has come a long way since Epstein Barr virus, the first human tumor virus, was identified from the cells of a patient with Burkitt's lymphoma in 1964. Those studies provide a framework for understanding how EBV perturbs cellular pathways, contributing to the development of cancer.

Epstein Barr virus (EBV) is closely associated with the development of a vast number of human cancers. To develop a system for monitoring early cellular and viral events associated with EBV infection a self-recombining BAC containing 172-kb of the Epstein Barr virus genome BAC-EBV designated as MD1 BAC (Chen et al., 2005, J.Virology) was used to introduce an expression cassette of green fluorescent protein (GFP) by homologous recombination, and the resultant BAC clone, BAC-GFP-EBV was transfected into the HEK 293T epithelial cell line. The resulting recombinant GFP EBV was induced to produce progeny virus by chemical inducer from the stable HEK 293T BAC GFP EBV cell line and the virus was used to immortalize human primary B-cell as monitored by green fluorescence and outgrowth of the primary B cells. The infection, B-cell activation and cell proliferation due to GFP EBV was monitored by the expression of the B-cell surface antigens CD5, CD10, CD19, CD23, CD39, CD40 , CD44 and the intercellular proliferation marker Ki-67 using Flow cytometry. The results show a dramatic increase in Ki-67 which continues to increase by 6-7 days post-infection. Likewise, CD40 signals showed a gradual increase, whereas CD23 signals were increased by 6-12 hours, maximally by 3 days and then decreased. Monitoring the viral gene expression pattern showed an early burst of lytic gene expression. This up-regulation of lytic gene expression prior to latent genes during early infection strongly suggests that EBV infects primary B-cell with an initial burst of lytic gene expression and the resulting progeny virus is competent for infecting new primary B-cells. This process may be critical for establishment of latency prior to cellular transformation. The newly infected primary B-cells can be further analyzed for investigating B cell activation due to EBV infection.