Robert P. Thomas

Age-associated changes in gene expression patterns in the liver

Aging is one of the least clearly understood biological processes. Alteration of oxidation/reduction (redox) enzymes has been demonstrated with aging; however, a systematic analysis of expression patterns has not been performed. The liver plays a key role in homeostasis and detoxification; therefore alteration of hepatic gene expression with aging may affect outcome after surgery. The purpose of our study was to assess changes in gene expression patterns in aged livers from both rats and humans using gene array analysis. Total RNA was extracted from young (2-month-old) and aged (2-year-old) rat livers, as well as young (1-year-old) and aged (78-year-old) human livers. Gene expression patterns were compared using Affymetrix GeneChip arrays. The expression pattern of selected genes was confirmed by reverse transcription-polymerase chain reaction. A threefold or greater change in gene expression was noted in 582 genes in the aged rat livers and 192 genes in the aged human livers. Comparison of the genes that were increased with aging demonstrated some similar patterns of expression in the rat and human livers, particularly in members of the antioxidant family and the cytochrome P-450 genes. Our findings demonstrate changes in the expression pattern of genes in the liver with aging. Concomitant increases in the expression of important antioxidant and detoxifying genes were noted in the livers of both rats and humans. This induction pattern suggests a complex link between changing hepatic detoxification/redox capability and senescence.

Activation of conventional PKC isoforms increases expression of the pro-apoptotic protein bad and trail receptors

AbstractBackground. Pancreatic cancer is a leading cause of cancer death worldwide; current treatment options have been ineffective in prolonging survival. Agents that target specific signaling pathways (e.g., protein kinase C [PKC]) may regulate apoptotic gene expression rendering resistant cancers sensitive to the effects of other chemotherapeutic drugs. The purpose of our study was to assess the effect of PKC stimulation on apoptotic gene expression in pancreatic cancer cells. Methods. The human pancreatic cancer cell line, PANC-1, was treated with PKC-stimulating agents, phorbol 12-myristate 13-acetate (PMA) or bryostatin-1, and analyzed for expression of apoptosis-related genes. Results. Both PMA and bryostatin-1 induced expression of the pro-apoptotic gene Bad in a dosedependent fashion. The expression of Bad was blocked by the PKC inhibitors GF109203x, Gö6983, and Ro-31-8220, suggesting a role for the conventional isoforms of PKC. In addition, treatment with the MEK inhibitors PD98059 or UO126 reduced PMA-mediated induction of Bad gene expression. PMA also increased the expression of TRAIL receptors DR4 and DR5; this expression was inhibited by the PKC inhibitors GF109203x, Gö6983, and Ro-31-8220 and the MEK inhibitor UO126, suggesting a role for conventional PKC isoforms and MEK in the regulation of TRAIL receptor expression. Conclusions. PKC stimulation in PANC-1 cells increases expression of the pro-apoptotic gene Bad and the TRAIL receptors, DR4 and DR5, through both conventional PKC- and MEK-dependent pathways. Agents that stimulate PKC may sensitize pancreatic cancer cells to apoptosis and provide a potential adjuvant therapy for the treatment of chemoresistant pancreatic cancers.

Effect of aging on the adaptive and proliferative capacity of the small bowel.

Our society is aging at a rapid rate; the effects of aging on physiologic functions (e.g., small bowel adaptation) are poorly understood. The purpose of this study was to determine the ability of the aged small bowel mucosa to adapt after resection. Young (2-month-old) and aged (24-month-old) F344 rats underwent massive (70%) proximal small bowel resection (SBR) or sham operation; rats were killed at 9 or 16 days after surgery. The remnant small bowel and corresponding sham segments were harvested, weighed, and analyzed for DNA content and villus height. To determine whether the adaptive response after SBR could be enhanced, aged rats underwent SBR or sham operation and were treated with either neurotensin or saline solution (control). SBR resulted in adaptive hyperplasia in the remaining small bowel remnant in both young and aged rats at 9 and 16 days compared with sham animals. At 9 days, significant increases were noted in weight, villus height, and DNA content of the distal remnant in young and aged rats after SBR; the increases were similar in both young and aged rats. At 16 days, both young and aged rats displayed significant increases in remnant weight after SBR. Administration of neurotensin increased the weight of the remnant intestine in aged rats after SBR compared with saline treatment. Our findings demonstrate that aged small bowel mucosa exhibits a proliferative and adaptive capacity in response to SBR that was similar to that of the young animals. In addition, neurotensin administration enhanced the normal adaptive response of the small bowel in aged rats, providing further evidence that neurotensin may be therapeutically useful to augment mucosal regeneration in the early periods after massive SBR.

Effect of gut transposition on the expression of the endocrine gene neurotensin

Expression of the gene encoding neurotensin (NT/N) is developmentally regulated in the adult small bowel with maximal expression noted in the distal ileum; the mechanisms responsible for this strict spatial-specific expression pattern are not known. The purpose of this study was to determine whether NT/N expression is altered by ileojejunal transposition. Rats underwent either sham operation or ileojejunal transposition and were killed 2 months after operation. The transposed (either jejunum or ileum) and sham-operated segments of gut were removed, a portion was processed for histologic examination, and the remainder was extracted for total RNA and analyzed by ribonuclease protection using a rat NT/N probe. For comparison, expression of another gut endocrine gene, peptide YY, and an enterocyte-specific gene, sucrase-isomaltase (SI), was also determined. Expression of the gut endocrine genes, NT/N and peptide YY, were minimally affected by transposition of either the jejunum or ileum. In contrast, SI expression was markedly altered in both the transposed jejunum and ileum compared with corresponding sham gut segments. Expression of the NT/N gene is minimally altered after jejunoileal transposition despite marked adaptive and morphologic changes in the transposed segments. These findings provide further support that the strict pattern of NT/N expression is “imprinted” in the gut and maintained regardless of location along the cephalocaudal gut axis.

CHAPTER 337 – Tropic Effects of Gut Hormones in the Gastrointestinal Tract

The mucosa of the gastrointestinal (GI) tract is a complex and constantly renewing tissue that is characterized by rapid proliferation, differentiation, and subsequent apoptosis, followed by extrusion into the GI lumen. These events occur as GI luminal epithelial cells ascend the vertical axis of the microfolded crypts lining the GI tract. This process normally takes 3-8 days, depending on the species and the location along the GI tract. Numerous factors can contribute to growth of the GI mucosa. This chapter focuses specifically on the effects of GI hormones on the proliferation and repair of non-neoplastic tissues and on the receptors and signaling pathways that transmit signals from the cell surface to the nucleus. By definition, any agent that stimulates growth can beconsidered a growth factor; however, these growth-stimulating agents are usually divided into those produced by normal cells and thought to act locally to control proliferation and hormones that are thought to act at a distance. In a limited clinical trial, administration of GLP-2 improved intestinal energy absorption, decreased energy excretion, increased body weight and lean body mass, and enhanced urinary creatinine excretion in patients with short bowel syndrome. In addition, GI hormones may play a role in preventing the severe sequelae of chemotherapeutic agents on the intestinal mucosa. Both BBS and GLP-2 have been shown to prevent the severe mucosal inflammation associated with various chemotherapeutic agents.