Gli2 protein expression level is a feasible marker of ligand-dependent hedgehog activation in pancreatic neoplasms.

The hedgehog pathway is known to promote proliferation of pancreatic ductal adenocarcinoma (PDA) and has been shown to restrain tumor progression. To understand how hedgehog causes these effects, we sought to carefully examine protein expression of hedgehog signaling components during different tumor stages. Genetically engineered mice, Pdx1-Cre;LSL-KrasG12D and Pdx1-Cre;LSL-KrasG12D;p53lox/+, were utilized to model distinct phases of tumorigenesis, pancreatic intraepithelial neoplasm (PanIN) and PDA. Human pancreatic specimens of intraductal papillary mucinous neoplasm (IPMN) and PDA were also employed. PanIN and IPMN lesions highly express Sonic Hedgehog, at a level that is slightly higher than that observed in PDA. GLI2 protein is also expressed in both PanIN/IPMN and PDA. Although there was no difference in the nuclear staining, the cytoplasmic GLI2 level in PDA was modest in comparison to that in PanIN/IPMN. Hedgehog interacting protein was strongly expressed in the precursors, whereas the level in PDA was significantly attenuated. There were no differences in expression of Patched1 at early and late stages. Finally, a strong correlation between Sonic Hedgehog and GLI2 staining was found in both human and murine pancreatic tumors. The results indicate that the GLI2 protein level could serve as a feasible marker of ligand-dependent hedgehog activation in pancreatic neoplasms.

Roles of the Pas1 and Par2 genes in determination of the unique, intermediate susceptibility of BALB/cByJ mice to urethane-induction of lung carcinogenesis: differential effects on tumor multiplicity, size and Kras2 mutations.

The C3H/HeJ (C3H), A/J and BALB/cByJ (BALB) mouse strains are respectively resistant, sensitive and intermediate regarding the induction of lung tumors by urethane. The phenotypic difference between C3H and A/J is largely determined by the Pas1 (Pulmonary adenoma susceptibility 1) gene on chromosome 6, the A/J allele of which dominantly increases the tumor burden. We recently found that BALB mice possess a unique lung tumor resistance gene on chromosome 18, designated Par2 (Pulmonary adenoma resistance 2), which partially, but dominantly suppresses the sensitive phenotype of A/J mice (Oncogene 13: 1599-1604, 1996). It has, however, remained unclear why BALB mice carrying the Par2 gene are significantly more sensitive to urethane-induced lung carcinogenesis than C3H mice that have no dominant lung tumor resistance genes. In the present study, using (C3H x BALB)F1 x C3H backcross mice treated with urethane, we demonstrated that BALB mice possess the disease allele of the Pas1 gene despite their 15-fold more resistance relative to A/J mice (LOD = 22.6). The BALB Par2 allele only significantly reduced the mean lung tumor multiplicity (LOD = 4.4) in the backcross population carrying the BALB allele of Pas1, indicating that the intermediate BALB phenotype may at least in part be the result of interactions between these two dominant genes. While the BALB Pas1 allele increased both the mean multiplicity and size of lung tumors, the BALB Par2 allele affected only the mean tumor multiplicity, implying that they are involved in different stages of multi-step lung carcinogenesis. In addition, we found that 68% of lung tumors from the BALB Pas1-positive backcross mice contained activating point mutations of the Kras2 oncogene, tightly linked to the Pas1 locus, whereas these genetic alterations were absent in tumors from BALB Pas1-negative mice. The Par2 genotype exhibited no effect on this parameter. Since the activating point mutations were observed exclusively in the BALB allele as already reported with lung tumors in (C57BL/6J x BALB/cJ)F1 mice, BALB Pas1 or possibly Kras2 itself may confer selective growth advantage on the affected urethane-initiated lung lesions.

The selective reduction in PTPdelta expression in hepatomas.

The mRNA levels for receptor-like protein tyrosine phosphatases (PTPases), PTPalpha, PTPdelta, PTPgamma and LAR, were evaluated by Northern blot analysis in two types of chemically-induced rat primary hepatomas. In the four PTPases the PTPdelta mRNA was selectively reduced in these hepatoma tissues. It was also diminished in HepG2 hepatoblastoma cell line and in all of the poorly differentiated ascites hepatoma cells examined. PTPalpha, PTPgamma and LAR did not show such a characteristic decrease. This selective reduction in PTPdelta expression strongly suggests PTPdelta plays an important role in hepatocarcinogenesis, possibly as a tumor suppressor gene.

Expression of an IL-1 receptor antagonist during mouse hepatocarcinogenesis demonstrated by differential display analysis.

The differential display technique was applied for identification of genes that have altered expression in mouse hepatocellular carcinomas relative to normal liver. Three genes were identified. The IL-1 receptor antagonist (IL-1ra) was expressed in hepatocellular carcinomas, whereas the major urinary protein (MUP) and cytochrome P-450 naphthalene hydroxylase (cyp2F2) genes were down-regulated. Because IL-1ra is a natural antagonist of IL-1, and because the latter has been reported to suppress the growth of hepatic cells, we also studied the expression of IL-1ra in hepatocarcinogenesis. IL-1ra was immunohistochemically detected in tumor cells in approximately 70% of hepatocellular adenomas and carcinomas, whereas early preneoplastic hepatocytic foci, as well as normal hepatocytes surrounding the lesions, were negative. In addition, 20% of human hepatocellular carcinomas were also partly positive for IL-1ra. RT-PCR analysis demonstrated that mouse hepatic tumors contain both secreted and intracellular forms of IL-1ra. On the other hand, there were no differences in levels of IL-1alpha and IL-1beta between hepatic tumors and normal liver in mice, suggesting that the majority of tumors create a microenvironment that inhibits the actions of IL-1. Furthermore, IL-1ra-positive adenomas contained more proliferating cell nuclear antigen-positive cells than IL-1ra-negative adenomas, indicating a link with high proliferation activity, although this was no longer evident in carcinomas. The observed altered gene expression may be related to biological phenotypes of hepatic tumors, and IL-1ra in particular may positively influence tumor cell growth through its antagonism of IL-1.

Loss of imprinting of the insulin-like growth factor II gene in mouse hepatocellular carcinoma cell lines.

We investigated expression of insulin-like growth factor II (Igf2) in primary cultured hepatocytes, liver epithelial (LE) cell lines derived from normal hepatocytes, and hepatocellular carcinoma (HCC) cell lines from crosses between C3H/HeJ (C3H) and Mus musculus molossinus mice (MSM). Igf2 mRNA was detected by reverse transcriptase-polymerase chain reaction in primary cultured hepatocytes from 5 d after the start of cultivation and in all 12 LE and 16 HCC cell lines. Analysis of the untranslated region of Igf2 exon 6, which contains polymorphic CA repeats, revealed that 13 of the 16 HCC cell lines had biallelic expression, whereas monoallelic expression was retained in the primary cultured hepatocytes and all 12 LE cell lines. The Igf2 transcripts contained exons 1-3 in all the HCC cell lines but only exons 2 and 3 in cultures of hepatocytes and LE cell lines, indicating difference in promoter use. However, the biallelic HCC cell lines did not have larger amounts of Igf2 mRNA and protein than did the monoallelic lines, suggesting that loss of imprinting may not be directly related to the level of Igf2 expression.

Nitric oxide release from the liver surface to the intraabdominal cavity during acute endotoxemia in rats.

Nitric oxide (NO) is produced by the liver during lipopolysaccharide (LPS)-induced endotoxemia. The aim of this study was to examine whether NO, which is produced in the liver, is released from the liver surface to the intraabdominal cavity during endotoxemia. NO was quantitatively determined by chemiluminescence and a newly developed gas purge technique was used to directly measure NO released from the liver surface and the intraabdominal cavity of rats before and after LPS (0.1 mg/kg, intraperitoneally) or saline administration. The expression of inducible NO synthase (iNOS) mRNA in the liver was detected by Northern blot analysis. NO levels from both the liver surface and in the intraabdominal cavity were elevated at 2 h after LPS injection and peaked at 10 h and both the time course of NO level were well correlated with each other. Both NO levels were below the detectable range before LPS and after saline administration. Inducible NOS mRNA in the liver exhibited a sharp increase to a maximum level at 4 h after LPS injection. The present study indicates that the hepatic NO, which might have been produced by iNOS in the liver, is released from the liver surface to the intraabdominal cavity during endotoxemia.

The characteristic gene expressions of MAPK phosphatases 1 and 2 in hepatocarcinogenesis, rat ascites hepatoma cells, and regenerating rat liver.

mRNA levels of mitogen-activated protein kinase phosphatases, MKP-1 and MKP-2, were determined during chemical hepatocarcinogenesis and during regeneration of rat liver. In chemical hepatocarcinogenesis, the mRNA levels of MKP-1 were increased in primary hepatomas but decreased in rat ascites hepatomas as compared with normal liver. MKP-2 was undetectable in normal liver but strongly expressed in hepatomas. The MKP-2 mRNA level was increased with expression of malignant phenotypes in hepatomas. In regenerating liver, the mRNA level of MKP-1 increased immediately but transiently after partial hepatectomy, and peaked again on day 10, the time when hepatocytes cease proliferation. The elevated expression of MKP-1 on day 10 suggests some roles of MKP-1 as a negative regulator in hepatocyte proliferation.