p53-mediated negative regulation of stathmin/Op18 expression is associated with G(2)/M cell-cycle arrest.

Utilizing the technique of differential display of mRNA, we have identified p53-responsive genes that are transcriptionally up- or down-regulated as cells enter growth arrest. One gene that was down-regulated, pong16, was found to be identical to stathmin/Op18, a protein involved in the regulation of microtubule dynamics. Evidence that p53 is directly or indirectly involved in negative regulation of stathmin/Op18 expression includes the following: (i) p53-mediated growth inhibition is associated with repression of stathmin/Op18 expression following serum stimulation, (ii) reporter gene assays revealed p53-mediated repression of stathmin/Op18 promoter activity and (iii) constitutive over-expression of stathmin/Op18 bypasses a p53-mediated G(2)/M arrest in the cell cycle. These results suggest that p53-mediated negative regulation of stathmin/Op18 plays an important role in cell-cycle control.

p53 mutants have selective dominant-negative effects on apoptosis but not growth arrest in human cancer cell lines.

A bidirectional expression vector that allowed equal transcription of cloned wild-type and mutant p53 cDNAs from the same vector was developed. The vector was transfected into CaLu 6 lung carcinoma cells or Saos-2 osteosarcoma cells. All p53 mutants examined were recessive to wild-type p53 transactivation of p21(WAF1/CIP1) but dominant-negative for transactivation of Bax. An examination of effects on growth arrest and apoptotic pathways indicated that all mutants were recessive to wild type for growth arrest but only three of seven mutants were dominant negative for induction of apoptosis.

Germ-line-derived hinge domain p53 mutants have lost apoptotic but not cell cycle arrest functions.

The protein p53 is a critical tumor suppressor, as demonstrated by its frequent mutation in human cancers. Overexpression of the wild-type form of the p53 tumor suppressor gene in human cancer cell lines has been shown to lead to either cell cycle arrest or apoptosis. A study of two Li-Fraumeni syndrome-derived p53 hinge domain mutants shows that both mutants retain the ability to arrest cell growth but are significantly impaired for the induction of apoptosis in human p53-null cell lines. This indicates that the hinge domain may be important in the regulation of p53-dependent apoptosis.

Identification of the MN/CA9 protein as a reliable diagnostic biomarker of clear cell carcinoma of the kidney.

The MN/CA9 protein is a tumor-associated antigen that has been shown to have diagnostic utility in identifying cervical dysplasia and carcinoma. MN/CA9 expression is limited to very few normal tissues. We have now extended those observations to further investigate expression of the MN/CA9 protein in histological sections and fine-needle aspiration biopsy smears of normal kidney, benign renal cell lesions, all categories of renal cell carcinomas (clear/granular/spindle cell, chromophilic cell, chromophobic cell, and collecting duct cell RCCs), metastatic RCCs, and non-renal cell clear cell adenocarcinomas. We have found that high levels of MN/CA9 expression is seen in all primary RCCs, cystic RCCs, and metastatic RCCs, with the exception of two cases of the chromophobe cell type, which were MN/CA9 negative. Identical MN/CA9 immunostaining was also observed in the aspiration cytological smears. In contrast, all benign lesions, including pyelonephritis, renal cysts, adenomas, oncocytomas, and normal kidney, did not express the MN/CA9 protein. Thus, we conclude that MN/CA9 protein expression could serve as a valuable adjunct to the cytological and histological diagnosis of benign renal cysts versus cystic RCC, adenoma versus RCC, and oncocytoma versus granular cell RCC. Diffuse membraneous staining of all RCCs (with the exception of chromophobic cell RCC) suggests that MN/CA9 protein expression might have an important clinical utility in the early detection and treatment of RCC. Absence of MN/CA9 expression in non-renal cell clear cell adenocarcinoma also indicates that MN/CA9 protein expression may be used as a differential diagnostic biomarker of metastatic clear cell RCC.