Attenuated acute liver injury in mice by naked hepatocyte growth factor gene transfer into skeletal muscle with electroporation.

BACKGROUND: Hepatocyte growth factor (HGF) plays an essential role in hepatic development and regeneration, and shows proliferative and antiapoptotic activity in hepatocytes. AIMS: To establish an effective new method for HGF gene transfer in vivo and to investigate its effects in acute experimental liver injury. ANIMALS: Eight week old female mice were used. METHODS: Rat HGF gene in a modified pKSCX plasmid was transferred to the tibialis anterior muscle by electroporation using a pulse generator. Four days later, plasma HGF concentrations were determined by enzyme linked immunosorbent assay every two days for three weeks. To confirm the efficacy of electroporation, a plasmid bearing green fluorescence protein (GFP) was transferred similarly. Four days after electroporation, carbon tetrachloride (CCl(4)) was administered to mice to induce acute liver injury. Plasma alanine aminotransferase (ALT) activity was measured. Hepatic apoptosis was assessed by Hoechst 33258 staining and the TUNEL method. RESULTS: Fluorescence microscopy showed strong green fluorescence where the GFP gene had been transferred into muscle. In mice given the HGF gene, HGF in plasma was increased up to fourfold from pretreatment amounts, peaking 6-9 days after electroporation and quickly decreasing within three weeks. Compared with the group without HGF transfer, the percentage of apoptotic hepatocytes after CCl(4) intoxication was significantly lower, as was ALT activity. In addition, ALT activity normalised more rapidly in the HGF gene transfer group. CONCLUSIONS: Naked DNA injection and transfer by electroporation efficiently brings about HGF expression in vivo, which can attenuate acute liver injury.

Antisense oligonucleotides specific to mutated K-ras genes inhibit invasiveness of human pancreatic cancer cell lines.

BACKGROUND/AIMS: Point mutations of the K-ras gene are detected in > 90% of human pancreatic cancers and may play an important role in tumorigenesis. However, correlations between mutant K-ras and the invasive activity of the tumor have remained unclarified. METHODS: 17-merphosphorothioate antisense oligonucleotides targeting K-ras point mutations were transfected into three kinds of human pancreatic cancer cell lines (MIAPaCa-2, PANC-1, and BxPC-3), and the invasive activity was investigated using an in vitro chemoinvasion assay. RESULTS: Antisense oligonucleotides strongly inhibited the invasive activity of the cell lines with mutant K-ras genes (MIAPaCa-2, PANC-1), but not in that with a wild-type K-ras (BxPC-3). CONCLUSION: Antisense oligonucleotides specific to mutated K-ras genes inhibited the invasiveness of human pancreatic cancer cell lines. Specific antisense therapy to the point mutation of K-ras might be a new anticancer strategy for pancreatic cancer.

"Return trip" metastases from the liver to the pancreas in Syrian golden hamsters.

In 1889, Paget suggested that metastases resulted only when "seed" (certain tumor cells) and "soil" (specific affinity for certain organs) were matched. We report here, the results of implantation of a hamster nitrosamine-induced pancreatic cancer tissue into the liver in six animals and the resulting metastases to the "primary organ". Survival time and metastatic sites at necropsy were studied. Tumor growth in the primary implantation site was observed in 5 out of 6 hamsters (83.3%) that died of cancer. Survival time ranged from 82 to 199 days. Metastases were found in the splenic lobe of the pancreas in all 5 (100%) successfully-implanted animals. This homologous implantation model of "return trip" in metastasis may be a useful tool for further research about the process of cancer metastasis. Moreover, it may be useful especially in the assessment of the "seed and soil" hypothesis.

Homologous orthotopic implantation models of pancreatic ductal cancer in Syrian golden hamsters: which is better for metastasis research--cell implantation or tissue implantation?

With a nitrosamine induced hamster pancreatic cancer cell line (HaP-T1), survival time and metastatic rates were compared between orthotopic cell implantation (OCI; n = 5) and orthotopic tissue implantation (OTI; n = 5) models. All the tumors were palpable (100% tumor take) after 1 week in both groups. Hamsters in the OCI group survived 71 +/- 2.17 days (range, 69-75 days), and in the OTI group, 73.8 +/- 4.03 days (range, 58-80 days). After necropsy, spontaneous metastases were noted in 100% of the animals. Direct invasion to adjacent organs was observed in four animals, and liver metastases, in three in the OTI group, which were significantly higher compared with the OCI group. On the other hand, peritoneal dissemination was observed only in the OCI group. Other metastatic sites showed no significant difference between the groups. All the histologically noted metastases had K-ras point mutation confirmed by polymerase chain reaction-restriction fragment length polymorphism (PCR/RFLP) analysis. We conclude that the homologous OTI model may be more useful than the OCI model. The OTI model may contribute to the development of therapeutic strategies in the field of pancreatic cancer research because of the capacity for invasion to adjacent organs, higher liver metastatic rate, and similarity to the clinical picture of the disease.

Subcutaneously inoculated cells and implanted pancreatic cancer tissue show different patterns of metastases in Syrian golden hamsters.

CONTEXT: We studied behavior of the subcutaneously implanted pancreatic tumors and the process of metastasis using syngeneic Syrian golden hamsters. DESIGN: HaP-T1, a cell line derived from nitrosamine-induced pancreatic cancer in Syrian golden hamsters was used for this experiment. Thirty-five animals were divided into two groups: subcutaneous cell inoculation and subcutaneous tissue implantation. The tumor tissue was obtained from subcutaneously implanted cancer cells. One month after implantation, the tumors were resected and studied histopathologically. The animals were followed-up weekly by palpation of the peripheral lymph nodes in order to identify local recurrence. After death, necropsy was performed. Liver, lungs and pancreas specimens were taken for histopathogical study and detection of K-ras point mutation using the PCR/RFLP method. RESULTS: The mean survival time in the subcutaneous cell inoculation group was 151+/-17.5 days, and in the subcutaneous tissue implantation group was 137 +/-12.9 days. During the follow-up, 13 subcutaneously cell inoculated hamsters (86.7%) had right axillary lymph node metastasis while subcutaneously tissue implanted hamsters did not show any palpable lymph nodes. After necropsy, 10 of the 20 subcutaneously tissue implanted animals (50%) showed metastases in the lungs at the histopathological level. However, 16 of the 20 subcutaneously tissue implanted animals (80%) showed K-ras point mutation in the lung specimens. The lungs of the animals of the subcutaneous cell inoculation group did not show any metastases. No metastases were found in the liver or the pancreas in either group. CONCLUSION: This study suggests that homologous subcutaneous cell inoculation and subcutaneous tissue implantation models showed completely different patterns of metastasis. These models may aid further research to clarify the mechanisms of metastasis in pancreatic cancer.

Curative resection of orthotopically implanted pancreatic cancer in Syrian golden hamsters.

BACKGROUND: Homologous orthotopic implantation models of pancreatic ductal cancer in Syrian golden hamsters can mimic the clinical features of the disease in humans. However, there have been no studies attempting a surgical approach to this animal model. Our aim was to clarify the possibility of curative resection in these tumor-bearing animals. METHODS: A nitrosamine-induced pancreatic ductal adenocarcinoma cell line, HaP-T1, was used for the experiments. Hamsters underwent two surgeries: first, the cells or the tissue previously grown subcutaneously were implanted into the pancreas, and second, pancreatectomy and splenectomy were performed within the following 4 wk after implantation. Animals were examined until death, when necropsies were performed. Surgically resected and necropsied specimens were studied histopathologically and at the molecular level by detection of K-ras point mutation at codon 12. RESULTS: At the second surgery, the success of implantation was 100%, and the curative resection rate was 75%. After observation for 400 d, 5 of 12 hamsters, which underwent second surgery within 15 d, were still alive. They were sacrificed and were found to be tumor-free. CONCLUSION: Orthotopic homologous implantation model of hamster pancreatic cancer combined with surgical resection may be useful for further research of adjuvant or neoadjuvant therapy for pancreatic carcinoma.

Growth inhibition of human pancreatic cancer cell lines by anti-sense oligonucleotides specific to mutated K-ras genes.

About 90% of human pancreatic cancers carry K-ras point mutation, which may play an important role in tumorigenesis. We investigated the inhibitory effects of anti-sense oligonucleotides targeting K-ras point mutation on the growth of cultured human pancreatic cancer cells. Eight human pancreatic cancer cell lines were screened for K-ras codon 12 point mutations by PCR-RFLP analysis and direct sequencing. Then, 3 cell lines with the major types of K-ras point mutation, i.e.,HuP-T1, HuP-T3 and PANC-1, and 1 without mutation, BxPC-3, were used for the experiments. Seventeen mer anti-sense oligonucleotides were designed, targeting the point mutation of K-ras codon 12, and transfected into the cells by the liposome-mediated method. Cell-growth activities were estimated by MTT assay. Levels of K-ras mRNA expression were determined using quantitative RT-PCR, and K-ras p21 protein synthesis was evaluated with Western blotting. Mutation-matched anti-sense oligonucleotides effectively inhibited the growth of these pancreatic cancer cell lines, except for BxPC-3, by suppressing K-ras mRNA expression and K-ras p21 protein synthesis. Moreover, mutation-matched anti-sense oligonucleotides showed stronger anti-proliferative effects than did mutation-mismatched ones. Our results suggest that anti-sense therapy specific to point mutations of K-ras mRNA is a practical approach to selective suppression of tumor growth, with little effect on normal cells.

Rapid recovery from anorexia nervosa after a life-threatening episode with severe thrombocytopenia: report of three cases.

We report the cases of three patients with anorexia nervosa (AN) who each recovered rapidly after experiencing a life-threatening episode with severe thrombocytopenia. All three cases were the typical restricting-type of AN, occurring in adolescence. They refused to be admitted to a hospital until their general condition had been severely deteriorated. Their lowest platelet counts were 2.9, 4.6, and 2.3 x 10(4)/mm3, respectively. Apparent hemorrhagic tendencies, such as purpura, gingival and nasal bleeding, and gastrointestinal bleeding were observed. The bone marrow examination showed apparent hypoplasia in two patients. No evidence of disseminated intravascular coagulation or autoantibody to platelets was detected. The platelet counts recovered rapidly by water and nutritional supplementation. The recovery from the AN itself was excellent in all three patients without specific psychotherapy.