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Pradip Raychaudhuri

Publications and source records attributed to Pradip Raychaudhuri.

16 recordsLinked to original sources
PubMed2006

Chk2 mediates stabilization of the FoxM1 transcription factor to stimulate expression of DNA repair genes.

The forkhead box M1 (FoxM1) transcription factor regulates expression of cell cycle genes essential for DNA replication and mitosis during organ repair and cancer progression. Here, we demonstrate that FoxM1-deficient (-/-) mouse embryonic fibroblasts and osteosarcoma U2OS cells depleted in FoxM1 levels by small interfering RNA transfection display increased DNA breaks, as evidenced by immunofluorescence focus staining for phosphospecific histone H2AX. FoxM1-deficient cells also exhibit stimulation of p53 transcriptional activity, as evidenced by increased expression of the p21(cip1) gene. FoxM1-deficient cells display reduced expression of the base excision repair factor X-ray cross-complementing group 1 (XRCC1) and breast cancer-associated gene 2 (BRCA2), the latter of which is involved in homologous recombination repair of DNA double-strand breaks. Furthermore, FoxM1 protein is phosphorylated by checkpoint kinase 2 (Chk2) in response to DNA damage. This phosphorylation of FoxM1 on serine residue 361 caused increased stability of the FoxM1 protein with corresponding increased transcription of XRCC1 and BRCA2 genes, both of which are required for repair of DNA damage. These results identify a novel role for FoxM1 in the transcriptional response during DNA damage/checkpoint signaling and show a novel mechanism by which Chk2 protein regulates expression of DNA repair enzymes.

Amino Acid Sequence↗
PubMed2006

A mutant allele of BARA/LIN-9 rescues the cdk4-/- phenotype by releasing the repression on E2F-regulated genes.

It has been proposed that C. elegans LIN-9 functions downstream of CDK4 in a pathway that regulates cell proliferation. Here, we report that mammalian BARA/LIN-9 is a predominantly nuclear protein that inhibits cell proliferation. More importantly, we demonstrate that BARA/LIN-9 also acts downstream of cyclin D/CDK4 in mammalian cells since (i) its antiproliferative effect is partially blocked by coexpression of cyclin D1, and (ii) a mutant form that lacks the first 84 amino acids rescues several phenotypic alterations observed in mice null for cdk4. Interestingly, mutation of BARA/LIN-9 restores the expression of E2F target genes in CDK4 null MEFs, indicating that the wild-type protein plays a role in the expression of genes required for the G1/S transition.

Alleles↗
PubMed2006

Cul4A and DDB1 associate with Skp2 to target p27Kip1 for proteolysis involving the COP9 signalosome.

DDB1, a subunit of the damaged-DNA binding protein DDB, has been shown to function also as an adaptor for Cul4A, a member of the cullin family of E3 ubiquitin ligase. The Cul4A-DDB1 complex remains associated with the COP9 signalosome, and that interaction is conserved from fission yeast to human. Studies with fission yeast suggested a role of the Pcu4-Ddb1-signalosome complex in the proteolysis of the replication inhibitor Spd1. Here we provide evidence that the function of replication inhibitor proteolysis is conserved in the mammalian DDB1-Cul4A-signalosome complex. We show that small interfering RNA-mediated knockdown of DDB1, CSN1 (a subunit of the signalosome), and Cul4A in mammalian cells causes an accumulation of p27Kip1. Moreover, expression of DDB1 reduces the level of p27Kip1 by increasing its decay rate. The DDB1-induced proteolysis of p27Kip1 requires signalosome and Cul4A, because DDB1 failed to increase the decay rate of p27Kip1 in cells deficient in CSN1 or Cul4A. Surprisingly, the DDB1-induced proteolysis of p27Kip1 also involves Skp2, an F-box protein that allows targeting of p27Kip1 for ubiquitination by the Skp1-Cul1-F-box complex. Moreover, we provide evidence for a physical association between Cul4A, DDB1, and Skp2. We speculate that the F-box protein Skp2, in addition to utilizing Cul1-Skp1, utilizes Cul4A-DDB1 to induce proteolysis of p27Kip1.

COP9 Signalosome Complex↗
PubMed2005

Tumor-prone phenotype of the DDB2-deficient mice.

DDB2 is an essential subunit of the damaged-DNA recognition factor DDB, which is involved in global genomic repair in human cells. Moreover, DDB2 is mutated in the repair-deficiency disease xeroderma pigmentosum (Group E). Expression of DDB2 in human cells is induced by P53, BRCA1 and by ionizing radiation. The DDB2 protein associates with transcriptional activator and coactivator proteins. In addition, DDB2 in conjunction with DDB1 associates with cullin 4A and the Cop9/signalosome. We generated a mouse strain deficient for DDB2 (DDB2-/-). Consistent with the human disease (XP-E), the DDB2-/- mice were susceptible to UV-induced skin carcinogenesis. We observed a significant difference in the initial rate of cyclobutane pyrimidine dimer (CPD)-removal from the skin following UV irradiation. Also, the DDB2-deficient mice exhibited a significantly reduced life span compared to their wild-type littermates. Moreover, unlike other XP-deficient mice, the DDB2-deficient mice developed spontaneous malignant tumors at a high rate between the ages of 20 and 25 months. The observations suggest that, in addition to DNA repair, the other interactions of DDB2 are significant in its tumor suppression function.

Animals↗
PubMed2005

New and unexpected: forkhead meets ARF.

Recent genetic studies demonstrate that mice deficient in the forkhead box m1b (Foxm1b) transcription factor are highly resistant to developing hepatocellular carcinoma, which is among the most lethal cancers worldwide. In addition, the Foxm1b transcription factor was identified as a novel inhibitory target of the p19ARF tumor suppressor during early stages of liver tumorigenesis, but p19ARF expression is extinguished in hepatic tumors that develop at later stages. Structure-function studies demonstrate that amino acids 26-46 of the p19ARF protein are sufficient to bind Foxm1b and reduce Foxm1b transcriptional activity by targeting it to the nucleolus. A peptide containing amino acids 24-46 of p19ARF, which was modified to enhance cellular uptake, is an effective inhibitor of Foxm1b transcriptional activity and prevents Foxm1b stimulation of anchorage-independent growth of cells on soft agar. Thus, the p19ARF peptide is an effective inhibitor of Foxm1b and represents a potential therapy for hepatocellular carcinoma.

Animals↗
PubMed2005

ARF directly binds DP1: interaction with DP1 coincides with the G1 arrest function of ARF.

The tumor suppressor ARF inhibits cell growth in response to oncogenic stress in a p53-dependent manner. Also, there is an increasing appreciation of ARF's ability to inhibit cell growth via multiple p53-independent mechanisms, including its ability to regulate the E2F pathway. We have investigated the interaction between the tumor suppressor ARF and DP1, the DNA binding partner of the E2F family of factors (E2Fs). We show that ARF directly binds to DP1. Interestingly, binding of ARF to DP1 results in an inhibition of the interaction between DP1 and E2F1. Moreover, ARF regulates the association of DP1 with its target gene, as evidenced by a chromatin immunoprecipitation assay with the dhfr promoter. By analyzing a series of ARF mutants, we demonstrate a strong correlation between ARF's ability to regulate DP1 and its ability to cause cell cycle arrest. S-phase inhibition by ARF is preceded by an inhibition of the E2F-activated genes. Moreover, we provide evidence that ARF inhibits the E2F-activated genes independently of p53 and Mdm2. Also, the interaction between ARF and DP1 is enhanced during oncogenic stress and "culture shock." Taken together, our results show that DP1 is a critical direct target of ARF.

Animals↗
PubMed2004

Cul4A physically associates with MDM2 and participates in the proteolysis of p53.

The cullin 4A (Cul4A) gene is amplified and overexpressed in breast and hepatocellular carcinomas. Cul4A functions as an E3 ligase and participates in the proteolysis of several regulatory proteins through the ubiquitin-proteasome pathway. Here, we show that Cul4A associates with MDM2 and p53. Depletion of Cul4A leads to an accumulation of p53. Moreover, expression of Cul4A increases the decay-rate of p53 and delays the accumulation of p53 in response to DNA damage. Cul4A fails to increase the decay of p53 in mouse embryonic fibroblasts lacking MDM2. In addition, the Cul4A-mediated rapid decay of p53 is blocked by p19ARF. The results provide evidence for a role of Cul4A in the MDM2-mediated proteolysis of p53.

Cullin Proteins↗
PubMed2004

Effects of depletion of CREB-binding protein on c-Myc regulation and cell cycle G1-S transition.

We recently reported that the transcriptional coactivator and histone acetyltransferase p300 plays an important role in the G(1) phase of the cell cycle by negatively regulating c-myc and thereby preventing premature G(1) exit (Kolli, et al. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 4646-4651; Baluchamy, et al. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 9524-9529). Because p300 does not substitute for all CREB-binding protein (CBP) functions, we investigated whether CBP also negatively regulates c-myc and prevents premature DNA synthesis. Here, we show that antisense-mediated depletion of CBP in serum-deprived human cells leads to induction of c-myc and that such cells emerge from quiescence without growth factors at a rate comparable with that of p300-depleted cells. The CBP-depleted cells contained significantly reduced levels of the cyclin-dependent kinase inhibitor p21 and low levels of p107 and p130 (but not pRb) phosphorylation, suggesting that these factors, along with elevated levels of c-Myc, contribute to induction of DNA synthesis. Antisense c-Myc reversed the phosphorylation of p107 and p130 and the induction of S phase in CBP-depleted cells, indicating that up-regulation of c-myc is directly responsible for the induction of S phase. Furthermore, the serum-stimulated p300/CBP-depleted cells did not traverse beyond S phase, and a significant number of these cells died of apoptosis, which was not related to p53 levels. These cells also contained significantly higher levels of c-Myc compared with normal cells. When c-myc expression was blocked by antisense c-Myc, the apoptosis of the serum-stimulated CBP-depleted cells was reversed, indicating that high levels of c-Myc contribute to apoptosis. Thus, despite their high degree of structural and functional similarities, normal levels of both p300 and CBP are essential for keeping c-myc in a repressed state in G(1) and thereby preventing inappropriate entry of cells into S phase. In addition, both these proteins also provide important functions in coordinated cell cycle progression.

CREB-Binding Protein↗
PubMed2004

Myc-ARF (alternate reading frame) interaction inhibits the functions of Myc.

The tumor suppressor protein ARF (alternate reading frame) inhibits MDM2 to stabilize and activate the functions of p53. Here we provide evidence for an additional activity of ARF that attenuates cell cycle progression independently of p53 activation. We show that ARF interacts with c-Myc independently of MDM2 or p53. Consequently, ARF relocalizes c-Myc from the nucleoplasm to the nucleolus. Binding and relocalization by ARF correlate with an inhibition of the c-Myc-activated transcription in both p53-positive and -negative cells. Using inducible cell lines, we show that the wild type ARF, but not a mutant, inhibits expression of the c-Myc-induced genes before inhibiting S phase. Moreover, ARF inhibits Myc-induced progression into S phase in cells lacking p53 or expressing a defective p53, indicating that ARF inhibits the S phase stimulatory function of c-Myc independently of p53. Our results strongly suggest that cMyc is a bona fide target of ARF and that ARF attenuates c-Myc independently of the ARF-p53 axis.

Alternative Splicing↗
PubMed2004

Foxm1b transcription factor is essential for development of hepatocellular carcinomas and is negatively regulated by the p19ARF tumor suppressor.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide. Here, we provide evidence that the Forkhead Box (Fox) m1b (Foxm1b or Foxm1) transcription factor is essential for the development of HCC. Conditionally deleted Foxm1b mouse hepatocytes fail to proliferate and are highly resistant to developing HCC in response to a Diethylnitrosamine (DEN)/Phenobarbital (PB) liver tumor-induction protocol. The mechanism of resistance to HCC development is associated with nuclear accumulation of the cell cycle inhibitor p27(Kip1) protein and reduced expression of the Cdk1-activator Cdc25B phosphatase. We showed that the Foxm1b transcription factor is a novel inhibitory target of the p19(ARF) tumor suppressor. Furthermore, we demonstrated that conditional overexpression of Foxm1b protein in osteosarcoma U2OS cells greatly enhances anchorage-independent growth of cell colonies on soft agar. A p19(ARF) 26-44 peptide containing nine D-Arg to enhance cellular uptake of the peptide was sufficient to significantly reduce both Foxm1b transcriptional activity and Foxm1b-induced growth of U2OS cell colonies on soft agar. These results suggest that this (D-Arg)(9)-p19(ARF) 26-44 peptide is a potential therapeutic inhibitor of Foxm1b function during cellular transformation. Our studies demonstrate that the Foxm1b transcription factor is required for proliferative expansion during tumor progression and constitutes a potential new target for therapy of human HCC tumors.

Adenoma↗
PubMed2003

Ddb1 is required for the proteolysis of the Schizosaccharomyces pombe replication inhibitor Spd1 during S phase and after DNA damage.

Recently we showed that the Schizosaccharomyces pombe ddb1 gene plays a role in S phase progression. A mutant S. pombe strain lacking expression of the ddb1 gene exhibited slow replication through both early and late regions causing a slow S phase phenotype. We attributed the phenotypes in the ddb1 strain to an increased activity of the replication checkpoint kinase Cds1. However, the basis for a high basal Cds1 activity in the ddb1 strain was not clear. It was shown that Ddb1 associates with the Cop9/signalosome. Moreover, the phenotypes of the Deltaddb1 strain are remarkably similar to the Deltacsn1 (or Deltacsn2) strain that lacks expression of the Csn1 (or Csn2) subunit of the Cop9/signalosome. Cop9/signalosome cooperates with Pcu4 to induce proteolysis of Spd1, which inhibits DNA replication by inhibiting ribonucleotide reductase. Therefore, we investigated whether Ddb1 is required for the proteolysis of Spd1. Here we show that a S. pombe strain lacking expression of Ddb1 fails to induce proteolysis of Spd1 in S phase and after DNA damage. Moreover, deletion of the spd1 gene attenuates the Cds1 kinase activity in cells lacking the expression of ddb1, suggesting that an accumulation of Spd1 results in the increase of Cds1 activity in the Deltaddb1 strain. In addition, the double mutant lacking spd1 and ddb1 no longer exhibits the growth defects and DNA damage sensitivity observed in the Deltaddb1 strain. Our results establish an essential role of Ddb1 in the proteolysis of Spd1. In addition, the observation provides evidence for a functional link between Ddb1 and the Cop9/signalosome.

Blotting, Western↗
PubMed2003

P19ARF inhibits the functions of the HPV16 E7 oncoprotein.

The E7 oncoprotein encoded by high-risk types of human papillomavirus (HPV) plays a significant role in the development of HPV-related cancers. E7 is a potent stimulator of S phase and host DNA replication. These functions of E7 are linked to the deregulation of the Rb family of proteins. For example, E7 binds and induces proteolysis of Rb through the ubiquitin-proteasome pathway. Despite advances in our understanding of E7, reagents that inhibit E7 with promise in therapy have not been developed or identified. Here, we provide evidence that the tumor suppressor ARF can inhibit E7. We show that the expression of ARF causes a relocalization of E7 from the nucleoplasm to the nucleolus. Two distinct regions in ARF overlapping with the MDM2-binding sites are necessary for the relocalization of E7. Furthermore, we show that ARF blocks the proteolysis of Rb induced by E7. In addition, ARF expression inhibits DNA replication induced by E7. Although it is not known whether the endogenous ARF, which is expressed at a low level, interferes with E7, our results suggest that ARF is an effective inhibitor of E7. We speculate that ARF or an ARF-derived molecule might have a significant impact in therapy against HPV-related tumors.

Cell Nucleolus↗
PubMed2003

Schizosaccharomyces pombe Ddb1 is functionally linked to the replication checkpoint pathway.

Schizosaccharomyces pombe Ddb1 is homologous to the mammalian DDB1 protein, which has been implicated in damaged-DNA recognition and global genomic repair. However, a recent study suggested that the S. pombe Ddb1 is involved in cell division and chromosomal segregation. Here, we provide evidence that the S. pombe Ddb1 is functionally linked to the replication checkpoint control gene cds1. We show that the S. pombe strain lacking ddb1 has slow growth due to delayed replication progression. Flow cytometric analysis shows an extensive heterogeneity in DNA content. Furthermore, the Deltaddb1 strain is hypersensitive to UV irradiation in S phase and is unable to tolerate a prolonged replication block imposed by hydroxyurea. Interestingly, the Deltaddb1 strain exhibits a high level of the Cds1 kinase activity during passage through S phase. Moreover, mutation of the cds1 gene relieves the defects observed in Deltaddb1 strain. The results suggest that many of the defects observed in Deltaddb1 cells are linked to an aberrant activation of Cds1, and that Ddb1 is functionally linked to Cds1.

Checkpoint Kinase 2↗
PubMed2003

Melphalan-induced up-regulation of B7-1 surface expression on normal splenic B cells.

We have previously shown that exposure of MOPC-315 or P815 tumor cells to the widely used anticancer drug melphalan ( L-PAM, L-phenylalanine mustard) leads to rapid up-regulation of B7-1 surface expression. Since B7-1-expressing tumor cells depend on B7-expressing host antigen presenting cells (APC) for the generation of CD8(+) T-cell-mediated antitumor immunity, and since L-PAM promotes the acquisition of tumor-eradicating immunity by CD8(+) T-cells from MOPC-315 tumor bearers, the current studies were undertaken to determine if L-PAM also up-regulates B7-1 expression on host APC. Here we show that exposure of normal spleen cells to L-PAM leads within 24 h to up-regulated B7-1 expression on B220(+) cells (B cells). Studies into the mechanism through which L-PAM leads to up-regulated B7-1 expression revealed that within 2 h after exposure of normal spleen cells to L-PAM, accumulation of B7-1 mRNA is evident and this accumulation requires de novo RNA synthesis, indicating that the regulation is at the transcriptional level. The L-PAM-induced accumulation of B7-1 mRNA was prevented with the antioxidant N-acetyl- L-cysteine (NAC), indicating that reactive oxygen species are important for the transcriptional regulation. Although AP-1 and NF-kappa B are considered redox-sensitive transcription factors, L-PAM led only to activation of NF-kappa B that bound specifically to a probe containing the corresponding binding site in the B7-1 gene. Moreover, selective inhibition of NF-kappa B activation prevented the L-PAM-induced B7-1 mRNA accumulation, indicating that NF-kappa B activation is essential for L-PAM-induced B7-1 gene expression in normal spleen cells. Finally, in vivo administration of an immunopotentiating dose of L-PAM to normal mice was found to up-regulate B7-1 mRNA expression in their spleens. Thus, the ability of L-PAM to up-regulate B7-1 expression not only on tumor cells but also on host cells may contribute to the potentiating activity of L-PAM for the acquisition of CD8(+) T-cell-mediated tumor-eradicating immunity in tumor bearers.

Animals↗
PubMed2002

Differential regulation of E2F1, DP1, and the E2F1/DP1 complex by ARF.

The tumor suppressor protein ARF inhibits MDM2 to activate and stabilize p53. Recent studies provided evidence for p53-independent tumor suppression functions of ARF. For example, it has been shown that ARF induces proteolysis of certain E2F species, including E2F1. In addition, ARF relocalizes E2F1 from the nucleoplasm to nucleolus and inhibits E2F1-activated transcription. Because DP1 is a functional partner of the E2F family of factors, we investigated whether DP1 is also regulated by ARF. Here we show that DP1 associates with ARF. Coexpression of ARF relocalizes DP1 from the cytoplasm to the nucleolus, suggesting that DP1 is also a target of the ARF regulatory pathways. Surprisingly, however, the E2F1/DP1 complex is refractory to ARF regulation. Coexpression of E2F1 and DP1 blocks ARF-induced relocalization of either subunit to the nucleolus. The E2F1/DP1 complex localizes in the nucleoplasm, whereas ARF is detected in the nucleolus, suggesting that ARF does not interact with the E2F1/DP1 complex. Moreover, we show that E2F1 is more stable in the presence of ARF when coexpressed with DP1. These results suggest that ARF differentially regulates the free and heterodimeric forms of E2F1 and DP1. DP1 is a constitutively expressed protein, whereas E2F1 is mainly expressed at the G(1)/S boundary of the cell cycle. Therefore, the E2F1/DP1 complex is abundant only between late G(1) and early S phase. Our results on the differential regulation E2F1, DP1, and the E2F1/DP1 complex suggest the possibility that ARF regulates the function of these cell cycle factors by altering the dynamics of their heterodimerization during progression from G(1) to S phase.

Animals↗