Phillip D. Gray

Conditional deletion of β1 integrins in the intestinal epithelium causes a loss of Hedgehog expression, intestinal hyperplasia, and early postnatal lethality

Conditional deletion of β1 integrins in the intestinal epithelium, unlike in epidermal and mammary epithelia, of mice does not result in decreased cell adhesion and proliferation, but instead causes a profound increase in epithelial proliferation with dysplasia and polypoid structures. The increased epithelial proliferation inhibited epithelial differentiation that caused severe malnutrition and early postnatal lethality. The striking similarities between β1 integrin–deleted mice and neonatal mice with defective Hedgehog signaling led to the discovery that Hedgehog expression was markedly reduced in the former mice. β1 integrins were found to drive the expression of Hedgehogs in intestinal epithelial cells in an HNF-3β (Foxa2)–dependent fashion. The expression of Tcf-4, a transcription factor known to be required for intestinal epithelial stem cell proliferation, was increased and mislocalized in the intestinal epithelia of the β1 integrin–deleted mice and in newborn mice treated with the Hedgehog signaling inhibitor cyclopamine. This study shows that β1 integrins are key regulators of proliferation and homeostasis in the intestine and achieve this not through anchorage-dependent effects but by generating Hh expression and signaling.

The Toxicity of Metabolites of Sodium Valproate in Cultured Hepatocytes

Abstract: Sodium valproate is hepatotoxic in both humans and rat hepatocytes. The toxicity is dose related and frequently associated with simultaneous ingestion of drugs which induce the drug metabolizing system. For these reasons, metabolites of sodium valproate were tested for toxicity using rat hepatocyte cultures. The sodium salts of three metabolites, 2‐propylpent‐4‐enoate, 4‐hydroxyvalproate, and perhaps 5‐hydroxyvalproate, were toxic in this system. In addition, 2‐propylpent‐4‐enoate was toxic in a dose‐related fashion. All are ω and ω‐1 oxidation products in the microsome‐mediated pathway of valproate metabolism.

The marine alkaloid naamidine A promotes caspase-dependent apoptosis in tumor cells

Apoptosis is important for normal development and removal of damaged cells. Evasion of apoptosis by cancer cells is one of the key characteristics of many tumor types. Thus, discovering agents that promote apoptosis in tumor cells could have great therapeutic value. Marine natural products have demonstrated great potential as anticancer agents, and the proapoptotic activity of some of these products is emerging as a potentially useful property for cancer treatments. Using a tumor xenograft assay in rodents, we previously found that the marine alkaloid naamidine A is a potent antitumor agent. In this study, we further characterize the mechanism of action of naamidine A. In cultured tumor cells, we find that naamidine A induces cell death, which is accompanied with annexin V staining, disruption of the mitochondrial membrane potential, and cleavage and activation of caspases 3, 8, and 9, all of which are hallmarks of apoptosis. Furthermore, naamidine A-induced cell death is caspase dependent. We also find that under conditions where naamidine A inhibits tumor xenograft growth, it induces activation of caspase 3, suggesting that apoptosis is part of its antitumorigenic activity in vivo. Apoptosis is not dependent on extracellular signal-regulated kinase 1/2, previously characterized molecular targets of naamidine A, nor does it require functional p53. Our studies support the continued study of naamidine A and its target(s) for the potential development of better clinical treatments for cancer.

Effect of Glycine on Valproate Toxicity in Rat Hepatocytes

Summary: The interaction between the amino acid glycine and valproate (VPA), an antiepileptic drug (AED) that occasionally causes hepatotoxicity, was studied in rat hepatocytes in monolayer culture. Valproate caused a doseependent increase in leakage of lactic acid dehy‐drogenase (LDH), and glycine prevented this toxic response. L‐Carnitine, L‐lanine, and L‐cysteine did not protect hepatocytes from VPA. Glycine also partially antagonized inhibition of fatty acid β‐oxidation by VPA, as estimated by the generation of acid‐soluble products from [14C]palmitic acid. These results are consistent with the hypothesis that glycine prevents VPA toxicity by removing acyl‐CoA esters, which accumulate during VPA exposure and interfere with fatty acid p‐oxidation. Glycine, however, also antagonized the toxic effects of acetaminophen on hepatocytes, although at higher concentrations than required to protect hepatocytes from VPA. Because the mechanism of toxicity of acetaminophen probably is different from that of VPA, a nonspecific cytoprotective effect may contribute to glycine antagonism of valproate toxicity. Our results emphasize the importance of glycine in protecting hepatocytes from noxious insult in general as well as from VPA in particular.

Toxicity of heparin in isolated rat hepatocytes.

The effect of heparin on isolated rat hepatocytes in monolayer culture was assessed to investigate the observed increase in serum aminotransferase activity in patients treated with heparin for thromboembolic disorders. Cells were treated with porcine intestinal mucosal heparin or beef lung heparin in concentrations ranging from 0.01 to 100 units/ml. Toxicity was evaluated based on cell damage or death measured by LDH release into the culture media as a fraction of total system LDH (LDH index). Toxicity appeared at concentrations between 1 and 10 units/ml (P less than 0.05). The uptake and binding of heparin by the hepatocyte were evaluated by addition of tritium-labeled heparin to the cultures. Sucrose gradient centrifugation with isolation of the liver plasma membranes (LPM) showed little membrane binding of heparin. The majority of intracellular heparin was located in the cytosol fraction. Heparin gains access to hepatocytes and causes a dose-related toxic effect resulting in cell damage and death. This investigation indicates that the increased serum aminotransferase concentrations seen with heparin treatment may be due to a direct hepatotoxic effect of heparin.

Pharmacologic downregulation of c‐FLIPL restores juxtacrine death receptor‐mediated apoptosis in cancer cells in a peritoneal carcinomatosis model

Metastasis occurs when circulating cancer cells implant in normal secondary tissues. Paradoxically, many cancer cells express death receptors while many normal tissues express the cognate death receptor ligands, suggesting that cancer cells possess mechanisms to inhibit death receptor signaling. Pharmacological restoration of juxtacrine‐mediated death receptor signaling could prevent cancer cells from implanting in normal tissues such as the peritoneum. The results showed that BAY 11‐7085 significantly inhibited peritoneal carcinomatosis in mice following the introduction of colon and pancreatic cancer cell lines into the intra‐abdominal cavity. Treatment with BAY 11‐7085 restored juxtacrine death receptor signaling during the adhesion of the cancer cells to mesothelial cells, which line the peritoneum. BAY 11‐7085 rapidly inhibited c‐FLIPL expression in colon and pancreatic cancer cell lines during adhesion to mesothelial cells. Pancreatic cancer cells sorted for high c‐FLIPL expression formed peritoneal implants much more readily than cells with low c‐FLIPL expression, and RNAi inhibition of c‐FLIPL in colon cancer cells dramatically reduced peritoneal implantation. This is a novel demonstration that the restoration of death receptor‐mediated apoptotic signaling in cancer cells through the pharmacological inhibition of c‐FLIPL can inhibit tumor implantation in a clinically relevant model of peritoneal carcinomatosis, a fatal disease. Pharmacological inhibitors of FLIP hold promise as a way to curtail cancer cell colonization of secondary tissues.

Toxicity of uricosuric diuretics in rat hepatocyte culture

Several aryloxyacetic acid diuretics have shown hepatotoxicity in humans, yet there continues to be interest in developing these compounds because of the uricosuric properties of some of them. This study was designed to test the utility of the hepatocyte monolayer culture as a model for studying these compounds. In addition, an attempt was made to define the structural components that are common to hepatotoxicity. Ticrynafen, indacrinone, ethacrynic acid and A-49816, an investigational compound, were found to be toxic in hepatocyte cultures; thus, with the exception of indacrinone, paralleling the experience in humans. The toxic compounds share a ketodichlorophenoxyacetic acid chemical structure. A-56234, an investigational uricosuric, was also found to be toxic in cultures but has not been demonstrated to be hepatotoxic in humans in limited clinical experience. It does not possess the ketodichlorophenoxyacetic acid structure proper but may be metabolized to a closely related structure. Furosemide, which does not have the ketodichlorophenoxyacetic acid structure, was not toxic in hepatocyte cultures and has not been hepatotoxic in humans. Thus, the structure common to the toxic compounds is ketodichlorophenoxyacetic acid or a closely related compound. The hepatocyte monolayer system appears to be a good model for demonstrating toxicity and, perhaps, for predicting toxicity of new compounds under development.