Steven R. Schwarze

Novel Pathways Associated with Bypassing Cellular Senescence in Human Prostate Epithelial Cells

Cellular senescence forms a barrier that inhibits the acquisition of an immortal phenotype, a critical feature in tumorigenesis. The inactivation of multiple pathways that positively regulate senescence are required for immortalization. To identify these pathways in an unbiased manner, we performed DNA microarray analyses to assess the expression of 20,000 genes in human prostate epithelial cells (HPECs) passaged to senescence. These gene expression patterns were then compared with those of HPECs immortalized with the humanPapillomavirus 16 E7 oncoprotein. Senescent cells display gene expression patterns that reflect their nonproliferative, differentiated phenotype and express secretory proteases and extracellular matrix components. A comparison of genes transcriptionally up-regulated in senescence to those in which expression is significantly down-regulated in immortalized HPECs identified three genes: the chemokine BRAK,DOC1, and a member of the insulin-like growth factor axis,IGFBP-3. Expression of these genes is found to be uniformly lost in human prostate cancer cell lines and xenografts, and previously, their inactivation was documented in tumor samples. Thus, these genes may function in novel pathways that regulate senescence and are inactivated during immortalization. These changes may be critical not only in allowing cells to bypass senescence in vitrobut in the progression of prostate cancer in vivo.

High levels of mitochondrial DNA deletions in skeletal muscle of old rhesus monkeys

Mitochondrial DNA (mtDNA) deletions increase in abundance with age in many tissues, however, their calculated low levels (usually < 0.1%) in samples from tissue homogenates containing thousands of cells argue against physiologic significance. Through the analysis of defined numbers of cells (skeletal muscle fibers) from rhesus monkeys, we report that the calculated abundance of specific mtDNA deletions is dependent upon the number of fibers analyzed: as the number of fibers decreases, the calculated deletion abundance increases. Also, most mtDNA deletions appear to occur in a mosaic pattern, varying from cell to cell in size, number and abundance. These data support the hypothesis that mtDNA deletions can focally accumulate to high levels contributing to declines in mass and function of aging skeletal muscle.

Oxidative stress and aging reduce COX I RNA and cytochrome oxidase activity in Drosophila

Drosophila melanogaster displays an age-associated increase in oxidative damage and a decrease in mitochondrial transcripts. To determine if these changes result in energy production deficiencies, we measured the electron transport system (ETS) enzyme activity, and ATP levels with age. No statistically significant influences of age on activities of complexes I and II or citrate synthase were observed. In contrast, from 2 to 45 days post-eclosion, declines were found in complex IV cytochrome c oxidase activity (COX, 40% decline) and ATP abundance (15%), while lipid peroxidation increased 71%. We next examined flies that were either genetically or chemically oxidatively stressed to determine the effect on levels of mitochondrial-encoded cytochrome oxidase I RNA (coxI) and COX activity. A catalase null mutant line had 48% of coxI RNA compared to the wild type. In Cu/Zn superoxide dismutase (cSOD) null flies, the rate of coxI RNA decline was greater than in controls. CoxI RNA also declined with increasing hydrogen peroxide (H2O2) treatment, which was reflected in reduced cytochrome c oxidase (COX) activity. These results show that oxidative stress is closely associated with reductions in mitochondrial transcript levels and support the hypothesis that oxidative stress may contribute to mitochondrial dysfunction and aging in D. melanogaster.

Role of cyclin-dependent kinase inhibitors in the growth arrest at senescence in human prostate epithelial and uroepithelial cells

Cellular senescence has been proposed to be an in vitro and in vivo block that cells must overcome in order to immortalize and become tumorigenic. To characterize these pathways, we focused on changes in the cyclin-dependent kinase inhibitors and their binding partners that underlie the cell cycle arrest at senescence. As a model, we utilized normal human prostate epithelial cell (HPEC) and human uroepithelial cell (HUC) cultures. After 30–40 population doublings cells became growth-arrested in G0/1 with a threefold decrease in Cdk2-associated activity, a point defined as pre-senescence. Temporally following this growth arrest, the cells develop a senescence morphology and express senescence-associated β-galactosidase (SA-β-gal). Levels of p16INK4a and p57KIP2 rise in HUCs during progressive passages, whereas only p16 increases in HPEC cultures. The induced expression of p57, similar to p16, produces a senescent-like phenotype. pRB, cyclin D, p19INK4d and p27KIP1 decrease in both cell types. We find that p53, p21CIP1 and p15INK4b are transiently elevated in HPECs and HUCs at the pre-senescent growth arrest, then return to low proliferating levels at terminal senescence. Analysis of p53, p21CIP1, p15INK4b, p16INK4a, and p57KIP2 reveals altered expression in immortalized, non-tumorigenic HPV16 E6 and E7 prostate lines and in tumorigenic prostate cancer cells. These results indicate: (i) the existence of a subset of growth inhibiting genes elevated at the onset of the senescence, (ii) a distinct class of genes involved in the maintenance of senescence, and (iii) the frequent inactivation of these pathways during immortalization.

Multiple age-associated mitochondrial DNA deletions in skeletal muscle of mice

Multiple mitochondrial DNA (mtDNA) deletions have been associated with aging in humans and monkeys. Since the inbred mouse strain, C57BL/6, has been extensively studied gerontologically, we sought to investigate its utility as a model for examining the importance of mtDNA deletions in aging. Using the polymerase chain reaction (PCR), we analyzed hind limb skeletal muscle from mice of three age groups (5, 16 and 25 months) for the presence of age- associated mtDNA deletions. We observed multiple mtDNA deletions in all three age groups. Further, the number of deletions detected per mouse increased greatly with advancing age. (Aging Clin. Exp. Res. 6: 193–200, 1994)

Alterations in the p16/pRb cell cycle checkpoint occur commonly in primary and metastatic human prostate cancer

We examined the status of a cell cycle checkpoint by immunohistochemically staining for p16 and pRb using multiple tissue arrays generated from 49 primary and 23 hormone-sensitive metastatic human prostate cancers. We find that p16, a cell cycle inhibitor, is paradoxically overexpressed in 83% of proliferating primary prostate cancers and increased expression correlates with a more rapid treatment failure (P=0.01) and a higher histologic grade (P=0.001). pRb staining is heterogeneous, loses expression infrequently (19%), and does not correlate with p16 expression. Loss of either p16 or pRb expression is found significantly (P=0.01) more commonly (55%) in metastatic specimens. The remarkable frequency of p16/pRb alterations and strong clinical associations implicates inactivation of this pathway as a critical determinant in prostate cancer progression.

Decreased mitochondrial RNA levels without accumulation of mitochondrial DNA deletions in aging Drosophila melanogaster.

Declines in electron transport system (ETS) activity have been reported to occur with advancing age in Drosophila melanogaster and many other animals. It has been proposed that these changes are importantly involved in the aging process. ETS decline has been attributed to mitochondrial nucleic acid damage. We analyzed various ages of D. melanogaster (embryos to 60-day-old adults) for the presence of mutated mitochondrial DNA (mtDNA) genomes. Although mtDNA genomes with large DNA deletions (up to 5 kb) were identified, abundance was low and remained constant throughout adult life. Therefore, these mtDNA deletions do not appear to be sufficiently abundant to cause large declines in ETS activity. Next, we analyzed various ages of D. melanogaster for the abundance of four mitochondrial-encoded and two nuclear-encoded ETS transcripts. The abundance of the mitochondrial transcripts declined 5-10-fold, while the nuclear-encoded transcripts declined 2-5-fold with advancing age. Separation of flies on the basis of flight loss was used to distinguish physiologic age from chronological age. Insects capable of flight at 30 days of age were found to have a 4-fold higher abundance of cox I mitochondrial-encoded RNA compared to flightless insects. No difference, however, was apparent in the nuclear-encoded beta-ATPase RNA level, suggesting only mitochondrial RNA (mtRNA) declines are associated with life expectancy.