Experimental and clinicopathologic studies on the function of the HGF receptor in human colon cancer metastasis

The organ-specific metastasis characterizes several human cancers, including colon carcinoma, a disease that frequently involves metastases in the liver. The data on the molecular mechanisms of liver metastasis would therefore be highly useful for prognostic purposes. Although the upregulation/amplification of the hepatocyte growth factor (HGF) receptor, c-met, has been frequently observed in colon cancer metastasis, the actual functional significance of the feature in the liver metastatization is not yet known. We have used three human colon carcinoma cell lines (HT29, HT25 and WiDr), characterized by different liver metastatic potentials in SCID mice, to analyze the expression of c-met and the biological effects of HGF. We found that HGF induces scattering in in vitro liver-metastatic cell lines (HT25 and WiDr) only at doses which are non-mitogenic (1–20 ng/ml). Analysis of the c-met expression revealed that the metastatic cell lines express authentic c-met gene and protein material, unlike the non-metastatic HT29 cell line, which expresses only the c-terminal cytoplasmic domain of the c-met β-chain. Interestingly, c-met was found to be localized in the substrate-attached peripheral membrane and partially colocalized with phosphotyrosine-proteins in the metastatic cells only when kept on fibronectin. On the other hand, we have analyzed 86 primary human colon cancers in Dukes' B (invasive but non-metastatic) and C (invasive and lymph node metastatic) stages. Western blotting of the proteins isolated from the tumor tissues and immunohistochemical control study on the paraffin samples of a third of these cases (25/86) all indicated a significant upregulation of the c-met protein in the Dukes' C tumor glands compared to the Dukes' B stages (P<0.001 and P<0.05, respectively). Since the two stages differ in the involvement of the regional lymph nodes but not in the invasion depth, the clinicopathological data and our experimental findings further support the notion that the c-met expression in human colon cancer can be considered as a marker of the metastatic potential due to its involvement in the generation of the motility signal. This revised version was published online in July 2006 with corrections to the Cover Date.     

Allelic losses on chromosome arm 10q and mutation of the PTEN (MMAC1) tumour suppressor gene in primary and metastatic malignant melanomas

Malignant melanomas frequently show loss of alleles on the long arm of chromosome 10. The PTEN (MMAC1) gene has been identified as a tumour suppressor gene at 10q23.3 that is mutated in various types of advanced human cancers. We have investigated a series of 40 sporadic melanomas from 37 patients (15 primary cutaneous melanomas and 25 melanoma metastases) for allelic losses on chromosome 10, as well as for deletion and mutation of the PTEN gene. Microsatellite analysis revealed loss of heterozygosity at loci located on 10q in tumours from 15 of 34 patients investigated (44%). Somatic PTEN mutations were identified in melanomas from 4 of 37 patients (11%), all of whom had metastatic disease. In two of these patients, the tumours had additionally lost one PTEN allele, indicating complete loss of wild-type PTEN in the tumour cells. Our findings corroborate that loss of heterozygosity on chromosome 10 is a frequent aberration in malignant melanomas and implicate PTEN as a tumour suppressor gene inactivated by somatic mutation in a fraction of these tumours. Received: 1 September 1999 / Accepted: 22 December 1999     

Effect of etoposide on experimental testicular teratoma in 129/SvJ mice

To study the effects of etoposide on experimental testicular teratoma in 129/SvJ mouse we analysed the tumour growth, differentiation, apoptosis and the localisation of mdr₁ P-glycoprotein (mdr₁-Pgp). In this model the implanted gonadal ridges developed into testicular teratomas in 17 out of 56 implanted testes (30%) and in 14 out of 28 mice (50%). The tumour-bearing mice were treated with etoposide on 4 successive days either 4 weeks or 6 weeks after implantation, and killed 7 days after the last dose. The mice in the control groups did not receive etoposide. The teratomas consisted mainly of neural tissue. The etoposide-treated 4-week teratomas, but not the 6-week teratomas, were significantly smaller than those in the corresponding control groups. The density of apoptotic cells and the distribution of the mdr₁-Pgp were not altered by etoposide. The decreased proportion of immature neuroectodermal tissue components was observed in all treated teratomas, converting the histology towards that of a mature teratoma. In addition, a low proportion of immature tissue components was frequently combined with a low density of apoptotic cells. In conclusion, etoposide decreased the immature tissue components of teratomas, while mature tissues remained unaffected. These results may have clinical relevance in man, since they confirm that postchemotherapy mature teratomas cannot be treated with chemotherapy. Despite benign histology, the human residual tumours have a significant malignant potential and require complete surgical excision and close surveillance. Received: 20 August 1999 / Accepted: 20 January 2000     

Longevity of immune complexes and abnormal germinal centre formation in NZB mice

Rheumatoid factor (RF)-like (antibody-antibody) immune complexes induce a selective and intensive immunoglobulin (Ig)G1-RF response after a single injection in mice. However, the longevity of the response differs between mouse strains: serum IgG1-RF antibody titres decline 40 days after injection in C57Bl/6 mice whereas levels are maintained for more than 100 days in NZB mice. In order to elucidate whether this difference was owing to a lower ability of NZB mice to clear the injected immune complexes, sections of kidney, spleen, liver and mesenteric lymph nodes were harvested at different time points after injection with RF-like immune complexes. Immunohistochemical staining revealed that NZB mice have a delayed clearance of the injected immune complexes, because the immune complexes are retained for more than 40 days in their spleens and 100 days in their kidneys, compared to only 14 days in C57Bl/6 mice. Germinal centres were also present for a longer period in the spleens of the NZB mice, accompanying the presence of the immune complexes, and were abnormally large compared to C57Bl/6 mice. The clearance of immune complexes from the spleen coincided with the decline in serum levels of IgG1-RF, indicating that prolonged retention of immune complexes is responsible for the sustained IgG1-RF response.     

Modes of adherence of Helicobacter pylori to gastric surface epithelium in gastroduodenal disease: a possible sequence of events leading to internalisation

We have investigated various modes of adherence of Helicobacter pylori to the human gastric epithelium, using transmission electron microscopy, in biopsies from nine patients with peptic ulcer disease and from four patients with chronic active gastritis. H. pylori was demonstrated in abundance in all cases within the surface mucous layer. In all ulcer- and in one out of four gastritis patients H. pylori was shown in close proximity to the gastric epithelium, with concurrent alterations in the configuration of microvilli and the apical cytoplasmic region of gastric cells. Previously described modes of H. pylori adherence were confirmed, such as loose attachment with fibrillar-like strands, firm attachment with pedestal formation, invasion in the intercellular spaces, and invagination with "cup" formation. Moreover, in many cases a fusion between the bacterial outer layer and gastric cell membranes was evident. In four cases (31%; three with active and one with past ulcer disease) viable H. pylori was found in the cytoplasm of gastric mucous cells. Our results support the hypothesis that the different modes of adherence of H. pylori represent a stepwise, possibly sequential, process which in a significant number of cases leads to internalisation of the organism. The invariable occurrence of adhesion and more frequent internalisation of H. pylori in ulcer patients may suggest a link with the pathogenesis of peptic ulcer disease.     

Effort reward imbalance is associated with vagal withdrawal in Danish public sector employees

Objectives

The current study analyzed the relationship between psychosocial work environment assessed by the Effort Reward Imbalance Model (ERI-model) and heart rate variability (HRV) measured at baseline and again, two years later, as this relationship is scarcely covered by the literature.

Methods

Measurements of HRV during seated rest were obtained from 231 public sector employees. The associations between the ERI-model, and HRV were examined using a series of mixed effects models. The dependent variables were the logarithmically transformed levels of HRV-measures. Gender and year of measurement were included as factors, whereas age, and time of measurement were included as covariates. Subject was included as a random effect.

Results

Effort and effort reward imbalance were positively associated with heart rate and the ratio between low frequency (LF) and high frequency power (HF) and negatively associated with total power (TP) and HF. Reward was positively associated with TP.

Conclusion

Adverse psychosocial work environment according to the ERI-model was associated with HRV, especially in the form of vagal withdrawal and most pronounced in women.