Aberrant methylation inactivates transforming growth factor Beta receptor I in head and neck squamous cell carcinoma.

Background. Alterations in TGF-beta signaling are common in head and neck cancer (HNSCC). Mutations in TGF-beta type II receptor (TbetaR-II) occur frequently in HNSCC while TGF-beta type I receptor (TbetaR-I) mutations are rare, suggesting that other molecular alterations in the TGF-beta pathway are likely. To identify abnormalities in TbetaR-I expression we analyzed 50 HNSCCs and correlated the results with clinical-pathologic features. Methods. Hypermethylation of TbetaR-I was evaluated via methylation-specific PCR (MSP) and restriction enzyme-mediated PCR (MSRE). Mutations in exons 1 and 7, mRNA and protein expression were analyzed by direct sequencing, semiquantitative RT-PCR and immunohistochemistry, respectively. Results. TbetaR-I expression was lost in 83% HNSCCs and was linked to DNA hypermethylation of the CpG-rich promoter region in 62% of the tumors. The variants 9A/6A and Int7G24A were found in two patients. Conclusions. This study shows that suppression of TbetaR-I expression in HNSCC is associated with DNA hypermethylation.

Differences in Smad4 expression in human papillomavirus type 16-positive and human papillomavirus type 16-negative head and neck squamous cell carcinoma.

The SMADs are a group of interrelated proteins that mediate transforming growth factor beta (TGF-beta) signaling. Upon TGF-beta binding the TGF-beta type I receptor phosphorylates Smad2 and Smad3, which then complex with Smad4 and translocate to the nucleus, with subsequent activation of target genes. Disruption of TGF-beta signaling is thought to contribute to the development of head and neck squamous cell carcinomas (HNSCC). Alterations in the function of the DPC4/Smad4 tumor suppressor gene have been found to inactivate TGF-beta signaling in several tumor types. For example, DPC4/Smad4 is lost or mutated in colorectal, pancreatic, and esophageal cancers. In addition, DPC4/Smad4 transcriptional activity and TGF-beta ability to inhibit DNA synthesis is blocked by the E7 protein of the human papillomavirus type 16 (HPV16) in cervical carcinoma cell lines. HPV16 infection is a risk factor for the development of a subset of HNSCC. This study was undertaken to investigate a potential correlation between expression of components of the TGF-beta signaling pathway and HPV16 status in HNSCC tumors. We examined the expression of TGF-beta signaling proteins Smad2, Smad2-P, and Smad4 by immunohistochemistry in 27 HPV16-negative and 16 HPV16-positive HNSCCs. We compared the expression patterns and assessed their relationship to HPV16 status. No significant differences were detected between HPV16-positive and HPV16-negative tumors in the expression of Smad2 and Smad2-P. Smad4 expression, however, was decreased in 56% of the HPV16-positive tumors and in 39% of HPV16-negative tumors. This difference was statistically significant (P = 0.01) suggesting that loss of Smad4 expression may be involved in HPV16-induced carcinogenesis of HNSCC.

Analytical and pharmacokinetic studies with 5-chloro-2'-deoxycytidine.

5-Chloro-2'-deoxycytidine (NSC 371331, CDC) is in development as a possible radiosensitizing agent for cancer treatment. Previous studies have been done to demonstrate the in vivo efficacy of CDC with various modulators of its metabolism. This paper describes our preclinical studies to determine the pharmacokinetic properties of CDC and the disposition of the drug, both alone and in the presence of the metabolic modulator tetrahydrouridine (THU), a cytidine deaminase inhibitor. Detection of the drug in biological fluids was performed by HPLC analysis using a C-18 column, gradient elution with solvents composed of aqueous trifluoroacetic acid and acetonitrile, and ultraviolet absorbance at 290 nm. Samples were processed by treatment with ammonium sulfate prior to injection into the HPLC system. CDC was stable in aqueous solution and in mouse plasma. High doses of CDC (100mg/kg) were given i.v. or i.p. to mice for the determination of CDC plasma half-life (10 min). CDC was not detectable in plasma after oral administration. It was converted rapidly to 5-chloro-2'-deoxyuridine (CDU) by cytidine deaminase, and CDU was readily discernable in plasma and urine samples collected after i.v. and i.p. administration of CDC. When CDC in doses ranging from 5 to 100mg/kg was given with 100mg/kg of THU, increased plasma levels of CDC were seen. CDC was eliminated through the kidneys, as well as by enzymatic deamination, and did not bind to plasma proteins. The initial steps of the CDC metabolic pathway were determined in vitro with isolated enzymes. Cytidine deaminase from mouse kidney converted CDC into CDU; thymidine phosphorylase converted CDU into 5-chlorouracil (5-CU). The conclusions of these studies are: (a) CDC is a drug with a short half-life and (b) it is excreted through the kidney, mainly in metabolite form. Administration of THU substantially increased the concentrations of CDC in mouse plasma, supporting proposals that the combination of THU with CDC should be evaluated in clinical trials.