Gian G. Re

p53/p21(WAF1/CIP1) expression and its possible role in G1 arrest and apoptosis in ellagic acid treated cancer cells

Ellagic acid is a phenolic compound present in fruits and nuts including raspberries, strawberries and walnuts. It is known to inhibit certain carcinogen-induced cancers and may have other chemopreventive properties. The effects of ellagic acid on cell cycle events and apoptosis were studied in cervical carcinoma (CaSki) cells. We found that ellagic acid at a concentration of 10(-5) M induced G arrest within 48 h, inhibited overall cell growth and induced apoptosis in CaSki cells after 72 h of treatment. Activation of the cdk inhibitory protein p21 by ellagic acid suggests a role for ellagic acid in cell cycle regulation of cancer cells.

Differential expression of genes induced by resveratrol in LNCAP cells: P53-mediated molecular targets

Prostate cancer prevention by key elements present in human nutrients derived from plants and fruits has been confirmed in various cell cultures and tumor models. Resveratrol (RE), a phytoalexin, induces remarkable inhibitory effects in prostate carcinogenesis via diverse cellular mechanisms associated with tumor initiation, promotion and progression. Earlier studies have shown that RE alters the expression of genes involved in cell cycle regulation and apoptosis, including cyclins, cdks, p53 and cdk inhibitors. However, most of the p53‐controlled effects related to the role of RE in transcription either by activation or repression of a sizable number of primary and secondary target genes have not been investigated. Our study examined whether RE activates a cascade of p53‐directed genes that are involved in apoptosis mechanism(s) or whether it modifies the androgen receptor and its co‐activators directly or indirectly and induces cell growth inhibition. We demonstrate by DNA microarray, RT‐PCR, Western blot and immunofluorescence analyses that treatment of androgen‐sensitive prostate cancer cells (LNCaP) with 10−5 M RE for 48 hr downregulates prostate‐specific antigen (PSA), AR co‐activator ARA 24 and NF‐kB p65. Altered expression of these genes is associated with an activation of p53‐responsive genes such as p53, PIG 7, p21Waf1‐Cip1, p300/CBP and Apaf‐1. The effect of RE on p300/CBP plays a central role in its cancer preventive mechanisms in LNCaP cells. Our results implicate activation of more than one set of functionally related molecular targets. At this point we have identified some of the key molecular targets associated with AR and p53 target genes. These findings point to the need for further extensive studies on AR co‐activators, such as p300, its central role in post‐translational modifications such as acetylation of p53 and/or AR by RE in a time‐ and dose‐dependent manner at different stages of prostate cancer that will fully elucidate the role of RE as a chemopreventive agent for prostate cancer in humans.

Identification of RPE65 in transformed kidney cells1

The protein RPE65 has an important role in retinoid processing and/or retinoid transport in the eye. Retinoids are involved in cell differentiation, embryogenesis and carcinogenesis. Since the kidney is known as an important site for retinoid metabolism, the expression of RPE65 in normal kidney and transformed kidney cells has been examined. The RPE65 mRNA was detected in transformed kidney cell lines including the human embryonic kidney cell line HEK293 and the African green monkey kidney cell lines COS‐1 and COS‐7 by reverse transcription PCR. In contrast, it was not detected in human primary kidney cells or monkey kidney tissues under the same PCR conditions. The RPE65 protein was also identified in COS‐7 and HEK293 cells by Western blot analysis using a monoclonal antibody to RPE65, but not in the primary kidney cells or kidney tissues. The RPE65 cDNA containing the full‐length encoding region was amplified from HEK293 and COS‐7 cells. DNA sequencing showed that the RPE65 cDNA from HEK293 cells is identical to the RPE65 cDNA from the human retinal pigment epithelium. The RPE65 from COS‐7 cells shares 98 and 99% sequence identity with human RPE65 at the nucleotide and amino acid levels, respectively. Moreover, the RPE65 mRNA was detected in three out of four renal tumor cultures analyzed including congenital mesoblastic nephroma and clear cell sarcoma of the kidney. These results demonstrated that transformed kidney cells express this retinoid processing protein, suggesting that these transformed cells may have an alternative retinoid metabolism not present in normal kidney cells.

Mitotic Chromosomal Bcl-2: II. Localization to Interphase Nuclei

We have previously shown, by immunofluorescence of fixed cells, that bcl-2 is found only in mitotic chromosomes in KB cultured human tumor cells expressing low levels of this oncoprotein. However, other studies showed that bcl-2 did not change its levels during the cell cycle when analyzed using Western blots. In this study we analyzed the distribution of bcl-2 during interphase, the point at which it is undetectable by immunofluorescence, using biochemical extraction, immunoprecipitation, and cell fractionation with Western blots. Interestingly, when carefully examined by immunofluorescence in fixed cells, the earliest point in the cell cycle showing bcl-2 localization was early G2, in which bcl-2 could be found within the intact nucleus. In spite of showing no immunofluorescence reaction in fixed interphase cells, immunoprecipitation of gentle detergent extracts showed that bcl-2 from interphase cells reacted readily with the antibody used (#124) after extraction. However, immunoprecipitation using anti-bcl-2 followed by Western blots using anti-Bax showed that, unlike overexpressing cells, this bcl-2 was not complexed with Bax. Classical cell fractionation methods were used to separate nuclei from cytosol and cell membranes. Surprisingly, these experiments clearly showed that essentially all of the bcl-2 in interphase KB cells was present in the nucleus. Therefore, the lack of reaction in fixed cells with anti-bcl-2 antibody reflects either a masking or a conformational change of the reactive epitope in bcl-2 present within the nucleus. By correlation, this change may be related to the phosphorylation of bcl-2 that occurs just before mitosis. The nature of this novel yet highly conserved nuclear form of bcl-2 and the understanding of its function will require further study.

Mitotic Chromosomal Bcl-2: I. Stable Expression Throughout the Cell Cycle and Association with Isolated Chromosomes

Bcl-2 is present in a cytoplasmic distribution in cells that express high levels of this oncoprotein. In contrast, using immunocytochemistry in cells expressing low levels of bcl-2, such as KB human carcinoma cells, we and others have shown that bcl-2 is present on the surface of mitotic chromosomes. However, monoclonal antibodies reactive with an epitope representing amino acids 41-54 of the bcl-2 sequence did not detect bcl-2 in other phases of the cell cycle. This study extended those earlier findings to determine if bcl-2 was expressed as a cyclin or if this pattern was an artifact of immunocytochemistry. Immunofluorescence studies in several other human cell lines showed the same mitotic distribution of bcl-2. Other studies using flow cytometry also showed selective mitotic phase detection of bcl-2. A comparison of available commercial antibodies showed that, in spite of reactivity with denatured bcl-2 on Western blots, clear reactivity with bcl-2 in fixed cells was found only with those reactive with the (a.a. 41-54) epitope. With RNase protection and Western blot analyses, cells synchronized at various stages of the cell cycle showed constant levels of bcl-2 mRNA and protein. Analysis of bcl-2 using Western blots showed a band with the same apparent molecular weight as that seen in comparison with authentic bcl-2 overexpressed in the cytoplasm. The retention of bcl-2 on chromosomes in unfixed, permeabilized preparations was influenced by protease treatment, phosphate, and pH. Studies using isolated chromosomes showed that much of the bcl-2 in these cells was attached to chromosomes in mitosis, had the expected molecular weight, and was phosphorylated in the same manner as that seen in whole-cell extracts. These results show that bcl-2 is not a cyclin and that the bcl-2 localized on chromosomes is the same molecule seen by immunoblotting. These results suggest that the reactive (a.a. 41-54) epitope present in bcl-2 is somehow modified or masked in interphase.

Induction of metallothionein mRNA and protein following exposure of cultured human proximal tubule cells to cadmium

Humans have a complex expression of metallothionein (MT) genes which involves many MT isoforms encoded by a family of genes containing an upper limit of 12 possible functional genes, in contrast to most animals which have one or two functional MT genes. In the present study, human proximal tubule (HPT) cells were exposed to cadmium (Cd) to determine if these cultures might serve as a model system to study MT gene expression in the renal proximal tubule. Three independent isolates of HPT cells were shown to repeatably induce MT protein when exposed continually to a non-toxic dose of 1 microgram/ml of Cd administered as CdCl2. Accumulation of MT protein was noted within 3 h and persisted over the 16-day time course. The expression of mRNA for the MT-IIA, MT-IA, B, E, F and G genes was also assessed through 16 days of exposure to 1 microgram/ml of Cd versus control media. Of these, the mRNA for the MT-IIA, MT-IE, MT-IF and MT-IG genes were detected in the cells exposed to 1 microgram/ml of Cd. Overall, the results were supportive that the HPT cells can provide a valuable model system to study the regulation of MT gene expression as it applies to the human renal proximal tubule.

Functional and gene expression analysis of the p53 signaling pathway in clear cell sarcoma of the kidney and congenital mesoblastic nephroma.

Mutation of p53 has been implicated in progression of classical Wilms tumor (WT) into the anaplastic variant (AWT), drug resistance, and poor prognosis. Because of prognostic similarities, clear cell sarcoma of the kidney (CCSK) has been classified with AWT and other aggressive pediatric renal tumors, apart from congenital mesoblastic nephroma (CMN), which is instead a relatively benign tumor of neonates. Initially, CCSK and CMN were assumed to be ontologically related, but the role of p53 in the pathogenesis of either disease has not been sufficiently evaluated as in AWT. We examined the status of p53 in CMN and CCSK in comparison to AWT by immunohistochemistry and mRNA analysis of p53, the downstream effector p21WAF-1/CIP-1 (p21), the multidrug resistance gene MDR-1, a putative target of p53, and the p53-antagonist Mdm-2. Surprisingly, strong p53 nuclear immunoreactivity was found in cultures from two CMN specimens, but not in frozen or fixed tumor tissue from five other CMN specimens, nor in cell lines or tumor tissue from CCSK. Sequence analysis excluded p53 mutations. The size of the p53 mRNA in CMN and CCSK primary tumors excluded gross deletions or rearrangements. Low levels of Mdm-2 mRNA in CCSK and CMN primary tumors and cultures did not support a role for Mdm-2. Absence of MDR-1 mRNA excluded MDR-1 in the drug-resistant phenotype of CCSK. Cisplatin-induced p21 transactivation assays and G1 cell cycle arrest analyses showed that p21 transactivation and G1 arrest occurred in both CCSK and CMN cultures, demonstrating integrity of the p53 signal transduction pathway. Absence of p53 functional abnormalities excluded relationships between CCSK and CMN as in AWT, supporting the association of cellular CMN with congenital fibrosarcomas as more recently proposed.