Mutations and Reduced Expression of the Transforming Growth Factor-β Receptor II Gene in Rat Lung Adenocarcinomas Induced by N-Nitrosobis-(2-hydroxypropyl)amine

Mutations and expression of the transforming growth factor-β receptor type II (TGF-βRII) gene were investigated in lung adenocarcinomas induced by N -nitrosobis(2-hydroxypropyl)amine (BHP) in rats. Males of the Wistar strain, 6 weeks old, were given 2000 ppm of BHP in their drinking water for 12 weeks and then maintained without further treatment until killed at week 25. Total RNA was extracted from 12 adenocarcinomas and mutations in TGF-βRII were investigated by RT-PCR-restriction-SSCP analysis followed by sequencing analysis. Two out of 12 adenocarcinomas showed band shifts, indicative of mutations (16.7%). One was a CTG-to-TTG (Leu to Leu) transition at codon 308 without amino acid alteration and the other a frameshift deletion of one of two guanines at nucleotides 1434 to 1435 (codon 477 to 478). Semi-quantitative RT-PCR analysis demonstrated significantly reduced TGF-βRII expression in adenocarcinomas, as compared with normal lung tissue. These results suggest that TGF-βRII alterations may play a role in the acquisition of growth advantage by lung adenocarcinomas induced by BHP in rats.     

Inhibition by Green Tea Extract of Diethylnitrosamine–initiated but Not Cholinedeficient, L–Amino Acid–defined Diet–associated Development of Putative Preneo–plastic, Glutathione S–Transferase Placental Form–positive Lesions in Rat Liver

The effects of green tea extract (GTE) on exogenous and endogenous models of rat liver carcinogenesis using diethylnitrosamine (DEN) and a choline–deficient, L–amino acid–defined (CDAA) diet were studied. For the exogenous carcinogenesis study, male Fischer 344 rats, 6 weeks old, were given a single intraperitoneal dose of 200 mg/Kg body weight of DEN, partially hepatectomized at week 3, and administered GTE at doses of 0, 0.01 and 0.1% in the drinking water from week 2 for 10 weeks. For the endogenous carcinogenesis study, rate were fed the CDAA diet and simultaneously given GTE for 12 weeks. All rats were killed at the end of week 12. After DEN–initiation, the apparent numbers of glutathione S–transferase placental form–positive foci, assayed as putative preneoplastic lesions, were decreased by the administration of GTE, though their sizes were not altered. In contrast, GTE did not significantly reduce the numbers of the lesions induced by the CDAA diet or affect their sizes. While the levels of 8–hydroxyguanine, a parameter of oxidative DNA damage, were reduced by the GTE administration in both experimental models, GTE did not protect against the CDAA–diet–associated liver tissue damage in terms of either histology or plasma marker enzyme levels. We conclude that, while GTE may be a possible chemopreventive agent for nitrosamine–initiated hepato–carcinogenesis in the absence of chronic hepatocyte damage, it does not significantly inhibit lesion development in hepatocarcinogenesis associated with the CDAA diet, a cirrhosis–associated model.     

A dominant negative mutation of transforming growth factor-beta receptor type II gene in microsatellite stable oesophageal carcinoma

Recent investigations revealed microsatellite instability in colon cancers are associated with mutations of the transforming growth factor-beta receptor type II gene (TGF-beta RII) that encodes a transmembrane protein containing an intracellular serine/threonine kinase domain. Activation of TGF-beta receptor type I (RI) and RII by TGF-beta induces nuclear translocation of Smad proteins including Smad2 and Smad4 that have been originally identified as tumour suppressor genes. We have previously reported six cases with microsatellite instability in 32 oesophageal carcinomas. In this study, we analysed genetic mutations of TGF-beta RII, Smad2 and Smad4 in these oesophageal carcinoma tissues and established 16 cell lines. No genetic mutation was detected in any tissues or cell lines except one tissue sample of microsatellite stable oesophageal carcinoma, that is, a mis-sense mutation of glutamic acid to glutamine at codon 526 (E526Q) in the TGF-beta RII serine/threonine kinase domain. Interestingly, the mutant TGF-beta RII E526Q can completely inhibit TGF-beta-induction of nuclear translocation of Smad4 protein in oesophageal carcinoma cells. This mutation of TGF-beta RII that is not associated with microsatellite instability might make a dominant negative effect on TGF-beta signal transduction in oesophageal carcinoma.     

Inhibition by N-(4-hydroxyphenyl)retinamide and all-trans-retinoic acid of exogenous and endogenous development of putative preneoplastic, glutathione S-transferase placental form-positive lesions in the livers of rats

The effects of N-(4-hydroxyphenyl)retinamide (4-HPR) and all-trans- retinoic acid (tRA) on the exogenous and endogenous models of rat liver carcinogenesis respectively using diethylnitrosamine (DEN) and a choline-deficient, L-amino acid-defined (CDAA) diet were studied. For the exogenous study, male Fischer 344 rats, 6 weeks old, were given a single i.p. dose of 200 mg/kg body wt of DEN, partially hepatectomized at week 3, administered 4-HPR at doses of 0, 0.04, 0.08 and 0.16% or tRA at 0, 0.004, 0.008 and 0.015% in diet from week 2 for 6 weeks, and killed at the end of week 8. For the endogenous study, rats were fed the CDAA diet containing 4-HPR or tRA for 12 weeks and killed at the end of week 12. 4-HPR decreased the numbers and sizes of the glutathione S-transferase placental form-positive foci, assayed as putative preneoplastic lesions, the levels of 8-hydroxyguanine (8-OHG), a parameter of oxidative DNA damage, and the bromodeoxyuridine labeling indices (BrdU L.I.) by all three doses in the DEN-initiated case and, more prominently, in the CDAA diet-associated case. In contrast, while tRA failed to exert inhibitory effects apparently on foci development, 8-OHG formation or BrdU labeling in the DEN-initiated case, it reduced the numbers and sizes of the foci, the 8-OHG levels and the BrdU L.I. by all three doses in the CDAA diet-associated case. Furthermore, both 4-HPR and tRA inhibited the CDAA diet-associated induction of hepatocyte necrosis and connective tissue increase but not intrahepatocellular fat accumulation. These results indicate that 4-HPR exerts chemopreventive effects against the exogenous and endogenous rat liver carcinogenesis, while tRA can inhibit only the latter.      

Frequent Microsatellite Instabilities and Analyses of the Related Genes in Familial Gastric Cancers

Microsatellite instability or replication error seems to be related to defective DNA mismatch repair genes, such as hMSH2 , hMLH1 , hPMS1 and hPMS2 , which have been identified as causative genes of hereditary nonpolyposis colorectal cancers (HNPCC). Recently, it was reported that mutations at the simple repeated sequences in the transforming growth factor-β type II receptor ( TGF-β RII ) gene occurred in replication error-positive colorectal cancers. To determine genetic alterations in familial gastric cancers (FGC), we examined replication error using eight microsatellite DNA markers, and screened mutations in the hMSH2 , hMLH1 and TGF-β RII genes in six cases from four FGC kindreds. Moreover, hMTH1 , a human homolog of the bacterial mutT gene, was also screened. Four of six (67%) cancers showed the replication error-positive phenotype, indicating that microsatellite instability is highly associated with not only HNPCC, but also FGC. No germline mutation was found in the whole coding sequences of hMSH2 and hMTH1 , or in the conservative regions of hMLH1 in any patient, while one cancer DNA showed a somatic mutation at codon 682 (threonine to alanine) in hMSH2 . No alteration was found at the small repeated sequences in TGF-β RII in FGC tumor DNA. These results indicate that the carcinogenetic process of FGC may be different from that of HNPCC.     

Analyses of the APC and TGF-β Type II Receptor Genes, and Microsatellite Instability in Mucosal Colorectal Carcinomas

APC and transforming growth factor-β type II receptor (TGF-β RII) gene mutations, and microsatcllitc instability have been found in sporadic colorectal carcinomas. To clarify further the early alterations in colorectal carcinogenesis, we investigated these genetic changes in 23 protruding- and 24 superficial-type mucosal colorectal carcinomas. TGF-β RII gene mutations and microsatellite instability were rarely found in these lesions. Nevertheless, APC was mutated in 16 of the 47 (34.0%) mucosal colorectal carcinomas and was significantly more frequently mutated in protruding- (I) and superficial elevated-type (Ila) (14/32,43.8%) than in other superficial-type (IIa+IIc, IIb, IIc, and IIc+IIa) (2/ 15,13.3%) mucosal colorectal carcinomas (P<0.04). These results indicate that the APC gene may be involved from the beginning in the tumorigenesis of many early colorectal carcinomas, particularly of the protruding and superficial elevated types. However, there might be a distinct pathway for other superficial-type colorectal carcinomas, possibly not involving APC as an initial step of tumorigenesis.     

Aberrant expression in multiple components of the transforming growth factor-β1-induced Smad signaling pathway during 7,12-dimethylbenz[a]anthracene-induced hamster buccal-pouch squamous-cell carcinogenesis

Transforming growth factor (TGF)-β1 signaling controls a plethora of cellular processes including tumorigenesis. The TGF-β1 ligand initiates signaling by binding to TGF-βreceptor II (TβRII) and allowing heterodimerization with TGF-βreceptor I (TβRI); thus, TβRI is phosphorylated by TβRII. After phosphorylation, Smad2 and Smad3 heterodimerize with Smad4, and this complex migrates to the nucleus to regulate the expression of specific target genes. However, Smad7 interrupts above signal transduction by preventing phosphorylation of Smad2 or Smad3. The objective of this study was to examine the TGF-β1-induced Smad signaling pathway during 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal-pouch squamous-cell carcinogenesis. Fifty 6-week-old male Syrian golden hamsters were divided into three experimental and two control groups (10 animals in each). Both pouches of each animal in the experimental groups were painted with 0.5% DMBA solution, and both pouches of each animal of one of the control groups were similarly treated with mineral oil; the other control group remained untreated throughout the experiment. Animals from three experimental groups were sacrificed at the end of 3rd, 9th, and 14th-weeks after DMBA treatment, respectively, and animals from two control groups were all sacrificed at 14th-weeks after the treatment. Immunohistochemical staining for TGF-β1, TβRI, TβRII, Smad2-4 and Smad7 were performed. RESULTS: A significant increase in the expression of Smad7 and significant decreases in the expression of TβRII, Smad 2, Smad3 and Smad4 were noted during hamster buccal-pouch carcinogenesis induced by DMBA. Our findings indicate that a disruption in TGF-β1-induced Smad signaling occurs as a result of aberrant expression of multiple components in the TGF-β1 signaling pathway during DMBA-induced hamster buccal-pouch carcinogenesis, leading to loss of TGF-β1 growth-suppressive effects on transformed pouch keratinocytes.     

Analysis of specific gene mutations in the transforming growth factor-beta signal transduction pathway in human ovarian cancer

Several proteins, including transforming growth factor beta (TGF-beta) receptor type I (RI), TGF-beta receptor type II (RII), Smad2, Smad3, and Smad4/DPC4, have been identified in the transduction pathway of the tumor suppressor TGF-beta. Mutations in TGF-beta RI, TGF-beta RII, Smad2, and Smad4/DPC4 genes are associated with several human cancers. The present study examines these gene mutations in 32 human ovarian cancers and 14 patient-matched normal tissues. For the first time, mutations in the Smad2 and Smad4 genes were analyzed in relation to human ovarian cancer. Gene mutations of TGF-beta RI, TGF-beta RII, Smad2, and Smad4 were analyzed using specific primers by PCR-single-strand conformational polymorphism (SSCP), and the results revealed a frameshift mutation at codons 276-277 (CTCTGG-->CTGCGTGG) in exon 5 of TGF-beta RI in 10 of 32 tumor samples (31.3%). This mutation was associated with reduced or absent expression of TGF-beta RI protein and p53 protein in tumor tissues. We detected SSCP variants of TGF-beta RII in exon 2 in 20 of 32 tumors. Sequence analysis of these variants revealed an A to G transition at the seventh band of intron 2. In this A to G polymorphism in intron 2, 12 samples (37.5%) had A/A alleles, 12 (37.5%) had A/G alleles, and 8 (25%) had G/G alleles. We detected Smad2 SSCP variants in exon 4 in 12 of 32 tumors (37.5%). Sequence analysis revealed a 2-bp deletion in the polypyrimidine tract of intron 3, which is located at position -39 to -56 in the splice acceptor site of the intron 3-exon 4 junction. No SSCP variants were detected in the Smad4 gene. These findings suggest that mutations in the TGF-beta RI and in its signal transduction pathway are likely responsible for human ovarian carcinogenesis.     

Clonal Heterogeneity in Human Esophageal Squamous Cell Carcinomas on DNA Analysis

Cancers are thought to arise through multistep accumulation of somatic mutations in the progeny of a single cell. Multiple mutations may induce molecular intratumor heterogeneity. Therefore, we examined molecular clonal heterogeneity in esophageal squamous cell carcinomas. Twenty-four esophageal squamous cell carcinomas and associated lymph node metastases were examined for microsatellite alterations, and abnormalities of the p53 and transforming growth factor-β type II receptor ( TGF-β RII ) genes. There were eight cases (33%) showing different patterns of loss of heterozygosity in primary tumors and metastatic lymph nodes with microsatellite markers. On the other hand, the abnormalities of p53 were identical in all these cases. No mutation was detected in the simple repeated sequences of the TGF-β RII gene. These results indicate that molecular clonal heterogeneity exists in esophageal squamous cell carcinomas. Therefore, care is necessary in preoperative genetic diagnosis using biopsy samples.     

c-Ski overexpression promotes tumor growth and angiogenesis through inhibition of transforming growth factor-β signaling in diffuse-type gastric carcinoma

c-Ski, originally identified as a proto-oncogene product, is an important negative regulator of transforming growth factor (TGF)-β family signaling through interaction with Smad2, Smad3, and Smad4. High expression of c-Ski has been found in some cancers, including gastric cancer. We previously showed that disruption of TGF-β signaling by dominant-negative TGF-β type II receptor in a diffuse-type gastric carcinoma model accelerated tumor growth through induction of tumor angiogenesis by decreased expression of the anti-angiogenic factor thrombospondin (TSP)-1. Here, we examined the function of c-Ski in human diffuse-type gastric carcinoma OCUM-2MLN cells. Overexpression of c-Ski inhibited TGF-β signaling in OCUM-2MLN cells. Interestingly, c-Ski overexpression resulted in extensive acceleration of the growth of subcutaneous xenografts in BALB/c nu/nu female mice (6 weeks of age). Similar to tumors expressing dominant-negative TGF-β type II receptor, histochemical studies revealed less fibrosis and increased angiogenesis in xenografted tumors expressing c-Ski compared to control tumors. Induction of TSP-1 mRNA by TGF-β was attenuated by c-Ski in vitro , and expression of TSP-1 mRNA was decreased in tumors expressing c-Ski in vivo . These findings suggest that c-Ski overexpression promotes the growth of diffuse-type gastric carcinoma through induction of angiogenesis. ( Cancer Sci  2009; 100: 1809–1816)