Inhibition of tumor cell growth by RTP/rit42 and its responsiveness to p53 and DNA damage.

Through a differential screening technique, we have identified a cDNA clone with differential expression in normal versus tumor cells. This clone, designated rit42 (reduced in tumor, 42 kDa), was previously isolated as a homocysteine-inducible gene in human endothelial cells (RTP), and the same or a highly related androgen-responsive gene in mouse has also been identified. Both Northern blot analysis and in situ hybridization demonstrated a significantly diminished expression in tumor cells, including those derived from breast and prostate when compared with normal cells. It was shown that RTP/rit42 mRNA cycles with cell division, peaking at G1 and G2-M, with lower expression in S phase. The biphasic expression of RTP/rit42 mRNA was absent in tumor cells. Introduction of rit42 cDNA into human cancer cells reduced cell growth both in vitro and in nude mice. Moreover, analysis of a tetracycline-regulated p53-inducible system in null-p53 cell lines showed that RTP/rit42 mRNA expression increased concomitantly with p53 expression and followed a similar time course. In addition, DNA-damaging agents induced RTP/rit42 expression in a p53-dependent manner but independent of a p53-mediated G1 arrest. Immunofluorescence analysis of a FLAG epitope-tagged RTP/rit42 protein revealed a cytoplasmic localization pattern with redistribution to the nucleus upon DNA damage. We have localized RTP/rit42 to human chromosome 8q24.3. Taken together, these results are consistent with a growth inhibitory role for RTP/rit42, and its down-regulation may contribute to the tumor malignant phenotype.

p21Waf1/Cip1/Sdi1 induces permanent growth arrest with markers of replicative senescence in human tumor cells lacking functional p53.

We have shown previously that wild type p53 can rapidly induce replicative senescence in EJ human bladder carcinoma cells lacking functional p53. A major effector of p53 functions is p21Waf1/Cip1/Sdi1, a potent cyclin-dependent kinase inhibitor. p21Waf1/Cip1/Sdi1 has been shown to be involved in both p53 dependent and independent control of cell proliferation, differentiation and death. To directly investigate the effects of p21Waf1/Cip1/Sdi1 in the p53 response observed in EJ tumor cells, we established p21Waf1/Cip1/Sdi1 inducible lines using the tetracycline-regulatable vector system. p21Waf1/Cip1/Sdi1 induction caused irreversible cell cycle arrest in both G1 and G2/M, and diminished Cdk2 kinase activity. In addition, p21Waf1/Cip1/Sdi1 induction led to morphological alterations characteristic of cells undergoing replicative senescence with morphological, biochemical and ultrastructural markers of the senescent phenotype. Furthermore, sustained p21Waf1/Cip1/Sdi1 induction sensitized EJ cells to apoptotic cell death induced by mitomycin C, a cross-linking DNA damaging agent. These findings support the function of p21Waf1/Cip1/Sdi1 as an inducer of replicative senescence and a major mediator of this phenomenon in response to p53. Moreover, our results imply that therapeutic intervention in human cancers might be aimed at sustained elevation of p21Waf1/Cip1/Sdi1 expression.

Glyceraldehyde-3-phosphate dehydrogenase in neurodegeneration and apoptosis signaling.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a well-studied glycolytic enzyme that plays a key role in energy metabolism. GAPDH catalyzes the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate in the glycolytic pathway. As part of the conversion, GAPDH converts NAD+ to the high-energy electron carrier NADH. GAPDH has been referred to as a "housekeeping" protein and based on the view that GAPDH gene expression remains constant under changing cellular conditions, the levels of GAPDH mRNA have frequently been used to normalize northern blots. In recent years, that view has changed since GAPDH is now known to contribute to a number of diverse cellular functions unrelated to glycolysis. Normative functions of GAPDH now include nuclear RNA export, DNA replication, DNA repair, exocytotic membrane fusion, cytoskeletal organization and phosphotransferase activity. Pathologically, GAPDH has been implicated in apoptosis, neurodegenerative disease, prostate cancer and viral pathogenesis (see Sirover (1999) for a recent review of GAPDH functions). Most recently, it has been shown that GAPDH is a target for deprenyl related compounds (Carlile et al., 2000; Kragten et al., 1998) and may contribute to the neuroprotection offered by those compounds.

Mitochondrial membrane potential in aging cells.

Decreased mitochondrial membrane potential (DeltaPsi(M)) has been found in a variety of aging cell types from several mammalian species. The physiological significance and mechanisms of the decreased DeltaPsi(M) in aging are not well understood. This review considers the generation of DeltaPsi(M) and its role in ATP generation together with factors that modify DeltaPsi(M) with emphasis on mitochondrial membrane permeability, particularly the role of a multiprotein membrane megapore, the mitochondrial permeability transition pore complex (PTPC). Previous data showing decreased DeltaPsi(M) in aged cells is considered in relation to the methods available to estimate DeltaPsi(M). In the past the majority of studies used whole cell rhodamine 123 fluorescence to estimate DeltaPsi(M) in lymphocytes from mice or rats. Imaging of DeltaPsi(M) in living, in situ mitochondria using laser confocal scanning microscopy offers advantages over whole cell measurements or those from isolated mitochondria, particularly if several different potentiometric dyes are employed. Furthermore, high resolution imaging of the newer fixable potentiometric dyes allows immunocytochemistry for specific proteins and DeltaPsi(M) to be examined in the same cells or even the same mitochondria. We found that decreased DeltaPsi(M) in p53 overexpression-induced or naturally occurring senescence is associated with decreased responsiveness of the PTPC to agents that induce either its opening or closing. The decreased PTPC responsiveness seems to reflect, at least in part, decreased levels of a key PTPC protein, the adenine nucleotide translocase. We also consider the possible basis for decreased DeltaPsi(M) in fibroblasts from patients with Parkinson's disease, an age-related neurodegenerative disease. Finally, we speculate on the mechanisms and functional significance of decreased DeltaPsi(M) in aging.

Wild-type p53 triggers a rapid senescence program in human tumor cells lacking functional p53.

The p53 tumor suppressor gene has been shown to play an important role in determining cell fate. Overexpression of wild-type p53 in tumor cells has been shown to lead to growth arrest or apoptosis. Previous studies in fibroblasts have provided indirect evidence for a link between p53 and senescence. Here we show, using an inducible p53 expression system, that wild-type p53 overexpression in EJ bladder carcinoma cells, which have lost functional p53, triggers the rapid onset of G1 and G2/M growth arrest associated with p21 up-regulation and repression of mitotic cyclins (cyclin A and B) and cdc2. Growth arrest in response to p53 induction became irreversible within 48-72 h, with cells exhibiting morphological features as well as specific biochemical and ultrastructural markers of the senescent phenotype. These findings provide direct evidence that p53 overexpression can activate the rapid onset of senescence in tumor cells.

Immunocytochemical localization of the neuron-specific form of the c-src gene product, pp60c-src(+), in rat brain.

Neurons express high levels of a variant form of the c-src gene product, denoted pp60c-src(+), which contains a 6 amino acid insert in the amino-terminal half of the c-src protein. We have determined the localization of pp60c-src(+) in neurons using an affinity-purified anti-peptide antibody, referred to as affi-SB12, that exclusively recognizes this neuron-specific form of the c-src gene product. Using affi-SB12, we examined the distribution of pp60c-src(+) by immunoperoxidase staining of sections through adult rat brains, pp60c-src(+) was widely distributed in rat brain and appeared to be differentially expressed in subpopulations of neurons. The majority of immunoreactive neurons was found in the mesencephalon, cerebellum, pons, and medulla. Telencephalic structures that contained substantial populations of pp60c-src(+)-immunoreactive neurons included layer V of the cerebral cortex and the ventral pallidum. Within individual neurons, pp60c-src(+) immunoreactivity was localized to the cell soma and dendritic processes, while labeling of axons and nerve terminals (puncta) was not as readily detected. Dense accumulations of immunoreactive axons were rare, being most prominent in portions of the inferior and superior olive, and in the spinal trigeminal nucleus. While the regional distribution of pp60c-src(+) immunoreactivity does not correlate with any specific neuronal cell type or first messenger system, this unique pattern of expression of pp60c-src(+) suggests the existence of a previously uncharacterized functional organization within the brain. Furthermore, the localization of this neuron-specific tyrosine kinase in functionally important areas of the nerve cell, namely, dendritic processes, axons, and nerve terminals, suggests that pp60c-src(+) may regulate pleiotropic functions in specific classes of neurons in the adult central nervous system.

Reduced mitochondrial membrane potential and altered responsiveness of a mitochondrial membrane megachannel in p53-induced senescence.

There is accumulating evidence that mitochondrial membrane potential (DeltaPsi(M)) is reduced in aged cells. In addition, a decrease of DeltaPsi(M) has been shown to be an early event in many forms of apoptosis. Here we use a mitochondrial potentiometric dye with in situ laser scanning confocal microscopic (LSCM) imaging to demonstrate that DeltaPsi(M) is dramatically decreased in both the p53-overexpressing, senescent EJ tumor cells and in pre-apoptotic PC12 cells compared to controls. Treatment with cyclosporin A (CSA), which facilitates closure of the mitochondrial permeability transition pore (PTP), was able to reverse the decrease in DeltaPsi(M) in pre-apoptotic PC12 cells but not in the senescent EJ-p53 cells. The capacity to prevent dissipation of DeltaPsi(M) in response to agents that facilitate PTP closure may differentiate cells entering apoptosis from those participating in senescence. Therefore, regulation of the closure of the mitochondrial PTP in the presence of decreased DeltaPsi(M) may be a decisional checkpoint in distinguishing between growth arrest pathways.