Combinatorial chemoprevention of intestinal neoplasia.

A combination of two drugs afforded remarkable protection from intestinal neoplasia in APC(Min/+) mice, a murine model of human familial adenomatous polyposis (FAP). One of the drugs was sulindac, a prototypical non-steroidal anti-inflammatory drug with established chemopreventative activity. The second drug was EKI-569, a newly developed, irreversible inhibitor of the epidermal growth factor receptor kinase. Although 100% of the untreated APC(Min/+) mice developed approximately 20 polyps, nearly half the mice treated with these two agents developed no polyps at all. These results suggest a powerful strategy for the chemoprevention of human colonic neoplasia.

Use of isogenic human cancer cells for high-throughput screening and drug discovery.

Cell-based screening for novel tumor-specific drugs has been compromised by the lack of appropriate control cells. We describe a strategy for drug screening based on isogenic human cancer cell lines in which key tumorigenic genes have been deleted by targeted homologous recombination. As a test case, a yellow fluorescent protein (YFP) expression vector was introduced into the colon cancer cell line DLD-1, and a blue fluorescent protein (BFP) expression vector was introduced into an isogenic derivative in which the mutant K-Ras allele had been deleted. Co-culture of both cell lines allowed facile screening for compounds with selective toxicity toward the mutant Ras genotype. Among 30,000 compounds screened, a novel cytidine nucleoside analog was identified that displayed selective activity in vitro and inhibited tumor xenografts containing mutant Ras. The present data demonstrate a broadly applicable approach for mining therapeutic agents targeted to the specific genetic alterations responsible for cancer development.

Effects of thyroid hormone on GLUT4 glucose transporter gene expression and NIDDM in rats.

Previous studies have shown that T3 coordinately stimulates GLUT4-glucose transporter messenger RNA (mRNA) and protein expression in mixed fiber-type skeletal muscle of the rat and produces a concomitant elevation in basal (noninsulin mediated) glucose uptake. The aim of the present study was to 1) determine the precise mechanism(s) for the T3-induced expression of GLUT4 in skeletal muscle, and 2) investigate the potential benefits of T3 on noninsulin dependent diabetes mellitus (NIDDM). Ten daily ip injections of T3 (100 micrograms/100 g BW) administered to hypothyroid male Sprague-Dawley rats, increased both GLUT4 mRNA and transcription approximately 70% (P < 0.05) in mixed fiber-type hindlimb skeletal muscle. Transcriptional induction was subsequently defined to be restricted to red (oxidative) muscle fibers (2.5-fold; P < 0.05), whereas GLUT4 protein was increased in both red and white (glycolytic) skeletal muscle. GLUT4 mRNA and protein expression were similarly inducible in the skeletal muscle of insulin-resistant Zucker rats. More importantly, T3 treatment totally ameliorated hyperinsulinemia in obese animals (P < 0.001), although their moderately elevated plasma glucose levels were not significantly altered. In conclusion, regulation of GLUT4 expression by T3 was shown to lie at the transcriptional level in red skeletal muscle, whereas in white muscle fiber types, it appears to operate via an alternative posttranscriptional mechanism. These data also support the potential of hormonally inducing glucose transporter expression in insulin-resistant muscle. However, high levels of T3 are associated with a number of adverse side-effects, in particular the stimulation of hepatic gluconeogenesis. Nevertheless, future studies may demonstrate, e.g. subthyrotoxic levels, to be similarly effective but without side effects, and thus perhaps find a clinical application in reducing both hyperinsulinemia and hyperglycemia in NIDDM.

Characterization of a low affinity thyroid hormone receptor binding site within the rat GLUT4 gene promoter.

Previous studies have demonstrated that thyroid hormone (T3) stimulates insulin-responsive glucose transporter (GLUT4) transcription and protein expression in rat skeletal muscle. The aim of the present study was to define a putative thyroid hormone response element (TRE) within the rat GLUT4 promoter and thus perhaps determine whether T3 acts directly to augment skeletal muscle GLUT4 transcription. To this end, electrophoretic mobility shift analyses were performed to analyze thyroid hormone receptor (TR) binding to a previously characterized 281-bp T3-responsive region of the rat GLUT4 promoter. Indeed, within this region, a TR-binding site of the standard DR + 4 TRE variety was located between bases -457/ -426 and was shown to posses a specific affinity for in vitro translated TRs. Interestingly, however, the GLUT4 TR-binding site demonstrated a significantly lower affinity compared to a consensus DR + 4 TRE, and only bound TRs appreciatively in the form of high affinity heterodimers, in this case with the cis-retinoic acid receptor. In conclusion, these data demonstrated the presence of a specific TR-binding site within a T3-responsive region of the rat GLUT4 promoter and thus support the supposition that thyroid hormone acts directly to stimulate GLUT4 transcription in rat skeletal muscle. Moreover, characterization of a novel TR-binding site with low affinity suggests an additional mechanism by which the intrinsic activity and responsiveness of thyroid hormone regulated genes may be modulated.

Gastrointestinal damage associated with the use of nonsteroidal antiinflammatory drugs.

BACKGROUND: Long-term use of nonsteroidal antiinflammatory drugs (NSAIDs) may lead to inflammation of the small intestine associated with occult blood and protein loss. The aim of this study was to investigate the prevalence and structural correlates of this enteropathy. METHODS: We examined the stomach, duodenum, and small intestine of 713 patients post mortem. Of these patients, 249 had had NSAIDs prescribed during the six months before death and 464 patients had not. All visible small intestinal lesions were removed for histologic examination, and specific etiologic factors were sought. The prevalence of nonspecific small-intestinal ulcers and ulcers of the stomach and duodenum was compared in the two groups of patients. RESULTS: Nonspecific small-intestinal ulceration was found in 21 (8.4 percent) of the users of NSAIDs and 3 (0.6 percent) of the nonusers (difference, 7.8 percent; 95 percent confidence interval, 5.0 to 10.6 percent; P less than 0.001). Three patients who were long-term users of NSAIDs were found to have died of perforated nonspecific small-intestinal ulcers. Ulcers of the stomach or duodenum were found in 54 (21.7 percent) of the patients who used these drugs and 57 (12.3 percent) of those who had not (difference, 9.4 percent; 95 percent confidence interval, 3.9 to 15.1 percent; P less than 0.001). CONCLUSIONS: Patients who take NSAIDs have an increased risk of nonspecific ulceration of the small-intestinal mucosa. These ulcers are less common than ulcers of the stomach or duodenum, but can lead to life-threatening complications.