Virchows Archiv

Rüschoff, J.; Bocker, T.; Schlegel, J.; Stumm, G.; Hofstaedter, F.

doi: 10.1007/BF00191357pmid: 7773499

Very recently a new molecular mechanism in the tumorigenesis of colorectal carcinoma has been described which is closely linked to hereditary non-polyposis colonic cancer (HNPCC). Ubiquitous changes in the length of simple repetitive DNA sequences between constitutional and tumour DNA occur in about 90% of cases of HNPCC and in about 15% of cases of non-familial, sporadic colorectal carcinoma. Such microsatellite instabilities have been shown to be the phenotypical marker of mutations in the human homologues of prokaryotic mismatch repair genes (MutS, MutL, MutH). These data provide crucial new tools in the detection of patients at high risk of developing colon cancer and other HNPCC-related carcinomas. In addition, these developments provide new insights into a new, presumably primary event in oncogenesis, i.e. the occurrence of mutations in genomic stability genes leading to an increased cellular mutation rate (“mutator phenotype”) and thus to cancer.

Schlegel, J.; Bocker, T.; Hofstädter, F.; Rüschoff, J.; Zirngabel, H.

doi: 10.1007/BF00191358pmid: 7773500

The aim of the present study was to establish a rapid, non-radioactive screening method for the detection of microsatellite instability (MIN). MIN is the primary characteristic of the mutator phenotype in tumours constituting hereditary non-polyposis colon cancers (HNPCC). We investigated 30 patients suffering from colorectal cancer using a non-radioactive PCR-based technique. MIN was present in 7 of 30 (23%) of the cases. There was a statistically significant correlation between MIN and localization of the tumour. Five of 7 (72%) tumours with MIN but only 4 of 23 (17%) tumours without MIN were localized in the proximal colon (P<0.01). There was a tendency to higher MIN frequency in tumours of patients with familial clustering of cancers. However, this was statistically not significant (P>0.05). In addition, no correlation between MIN and tumour grade and stage was found. For the investigations in the present study we used a non-radioactive PCR-based method followed by denaturating polyacrylamide gel electrophoresis and silver staining. This method is highly sensitive and reproducible. Thus, PCR-based analysis using a non-radioactive staining technique represents a comprehensive tool for MIN screening in diagnostic pathology.

Gorczyca, W.; Markiewski, M.; Kram, A.; Tuziak, T.; Domagala, W.

doi: 10.1007/BF00191359pmid: 7773501

We examined 59 breast cancers for p53 and bcl-2 protein expression by immunohistochemistry. The results were correlated with Ki-67 immunostaining. p53-negativity was noted in 40 cases and the remaining 19 tumours were p53-positive. Thirty-six tumours showed strong expression of bcl-2 and in 23 no staining for this protein was observed. We found statistically significant reverse correlation between expression of p53 and bcl-2 in majority of carcinomas: 31 cases were bcl-2 positive and p53-negative, and 14 tumours were bcl-2-negative and p53-positive. Six carcinomas showed no nuclear staining for Ki-67 and in the remaining 53 the percent of cancer cells positive for Ki-67 ranged from 1 to 60 (mean: 14.6). In these 53 cases we found that bcl-2-positive tumours were characterized by lower proliferation than bcl-2-negative tumours, the mean value of Ki-67 immunostaining being 10.7% and 23.0%, respectively. p53-negative tumours showed lower proliferation than p53-positive tumours: mean Ki-67 index was 10.2% and 23.9%, respectively.

Amling, M.; Werner, M.; Pösl, M.; Delling, G.; Maas, R.; Korn, U.

doi: 10.1007/BF00191360pmid: 7773502

Fourteen solitary bone cysts (SBC) with large areas of calcification (7 in the femur, 4 in the humerus, and 1 each in the pelvis, the tibia and the scapula) and 402 SBC from the Hamburg Bone Tumour Registry were reviewed in a retrospective study. The analysis was done with emphasis on the clinical, radiological and histological appearances. SBC are well known lesions, but calcifying SBC (CSBC) or extensive extragnathic cement-like bone productions are rare. The clinical and radiological differential diagnosis includes fibrous dysplasia, chondroma, low-grade chondrosarcoma and osteosarcoma. Bits of this cement-like matrix are detectable within the wall of approximately 70% (278 of 402) of SBC from the registry. CSBC are changed SBC. The intraoperative confirmation of the diagnosis on a frozen section by the bone pathologist leads to curettage which is currently the most common therapy in this benign lesion.

Kivistö, K.; Kroemer, H.; Linder, A.; Friedel, G.; Beaune, P.; Belloc, C.; Fritz, P.

doi: 10.1007/BF00191361pmid: 7773503

Cytochrome P450 2E1 (CYP2E1) is a major xenobiotic-metabolizing enzyme but data concerning its extrahepatic expression are few. CYP2E1 can metabolically activate many procarcinogens and therefore its presence in the lung might play a role in bioactivation of procarcinogens, so we studied the expression and localization of CYP2E1 in primary pulmonary carcinomas and surrounding normal bronchial tissue from 28 patients. Seromucous glands showed expression of CYP2E1 in 19 and bronchial epithelium in 18 of the 28 samples of normal bronchial tissue. Thirteen of the corresponding cases of primary pulmonary carcinoma showed staining for CYP2E1. In 11 of these 13 cases, CYP2E1 was also present in normal bronchial tissue. There was no statistically significant difference in the expression of CYP2E1 between adenocarcinomas and squamous cell carcinomas. No association was observed between the expression of CYP2E1 in tumour tissue and normal bronchial tissue. However, there was a significant correlation between the expression of CYP2E1 in seromucous glands and bronchial epithelium (r=0.61, P<0.01) of normal tissue. We conclude that CYP2E1 can be present in both normal and neoplastic bronchial tissue.

Seidel, A.; Bunge, A.; Schaefer, B.; Herzig, I.; Steidtmann, K.; Dietel, M.; Hasmann, M.; Löser, R.

doi: 10.1007/BF00191362pmid: 7773504

In the human gastric carcinoma cell line EPG85-257P (parent) induction of resistance to daunorubicin (DAU) was achieved by selection with stepwise increased concentrations of the drug. The new vairant was named EPG85-257DAU and was shown to overexpress the mdr1 gene product 170 kDa P-glycoprotein (P-Gp) as demonstrated by immunocytochemistry and mdr1-specific RT-PCR. To investigate the intracellular pathway of DAU the subcellular distribution of this autofluorescent drug was studied in the resistant cells and compared to its chemosensitive counterpart EPG85-257P. When sensitive cells were exposed to DAU the drug rapidly accumulated in the nucleus until cell death. No redistribution of DAU to the cytoplasm was observed. In resistant cells exposed to the drug DAU also accumulated in the nucleus but to a lesser extent than in parent cells. Following exposure, nuclear fluorescence was observed to decrease over a time period of up to 48 h. Six hours after DAU exposure formation of fluorescent vesicle formation started in the perinuclear region and increased continously. After 48 h nuclear fluorescence was no longer detectable and DAU was located exclusively in vesicles. During this period the vesicles moved from the region of origin to the cell periphery. A pulse chase experiment showed, that vesicles may contain DAU derived from the nucleus. Treatment of EPG85-257DAU cells with DAU in conjunction with the chemosensitizer cyclosporin A (CsA) increased nuclear fluorescence without impairing vesicle formation. Disruption of microtubules by nocodazole led to an accumulation of vesicles in the perinuclear region indicating that microtubules are involved in vesicular transport. Treatment of EPG85-257DAU cells with the actin disruptor cytochalasin B led to accumulation of vesicles in the cell periphery indicating that actin may be involved in exocytosis. Uptake and efflux of DAU and rhodamin (RH) were determined in sensitive and resistant cells using a fluorescence activated cell sorter. Uptake of both compounds was distinctly lower in resistant than in sensitive cells. When resistant cells preloaded for 2 h with RH subsequently were incubated in drug free medium the substance was rapidly released indicating transmembrane transport by P-Gp. In contrast, despite expression of P-Gp in resistant cells no considerable release of DAU was observed for up to 2 h under the same experimental protocol. This indicates that in resistant cells intracellular DAU at least in part may be inaccessible for P-Gp and that vesicular drug transport appears to contribute to DAU resistance by removing intracellular DAU via exocytosis.

Endresen, P.; Aarbakke, J.; Fandrem, J.; Eide, T.

doi: 10.1007/BF00191363pmid: 7773505

Using electron microscopy, confocal laser scanning microscopy and measurements of intact DNA we have studied the morphology and DNA degradation of human leukaemia HL-60 cells undergoing drug initiated apoptosis. Apoptosis was initiated by 100 μM 3-deazaadenosine (c3Ado), 25 μM c3Ado plus 1 mM homocysteine thiolactone (Hcy) and 100 μM c3Ado plus 5 μg/ml cytochalasin B (CB). Two different phenotypes of apoptotic cells (APC), blebbed and non-blebbed, were present in the cultures. Blebbed APC dominated in cultures exposed to c3Ado, whereas most APC in cultures treated with c3Ado plus Hcy and all the APC in cultures treated with c3Ado plus CB displayed a non-blebbed phenotype. A more pronounced reduction of the chromatin/cytoplasm ratio, lower volume fractions of uncondensed chromatin and higher volume fractions of highly condensed chromatin (micronuclei) were found in cultures exposed to c3Ado and c3Ado plus Hcy when compared with cultures exposed to c3Ado plus CB. A partial inhibition of c3Ado apoptosis by CB was confirmed by measurements of intact DNA. The inhibitory effect of CB was not reproducible by CE, indicating that CB exerts its effect by an actin independent mechanism. Both blebbed and non-blebbed APC displayed nuclear fragmentation, segregation of organelles and cytoplasmic vesiculation, suggesting that the differences between the phenotypes were restricted to the cytoplasmic membrane. We were not able to demonstrate the presence of F-actin by fluorescein isothiocyanate-phalloidin staining in blebbed APC nor in non-blebbed APC in cultures treated with c3Ado plus Hcy. Non-blebbed APC in cultures treated with c3Ado plus CB displayed foci of F-actin at the internal part of the cytoplasmic membrane. This suggests that F-actin is preserved by the mechanism by which CB inhibits blebbing, and may indicate that blebbing of the cytoplasmic membrane during apoptosis is associated with F-actin deficiency rather than a result of actin-myosin interactions.

Takayama, I.; Fujino, M.; Fujii, Y.; Ohno, S.

doi: 10.1007/BF00191364pmid: 7773506

Changes of intracellular localization of serotonin in rat mast cells were examined by freeze-fracture immunocytochemistry, to prevent the translocation of the serotonin antigen. Rat peritoneal cells including mast cells were stimulated in vitro with compound 48/80, at 17° C for 0, 30 or 60 s for exocytosis to occur. The mast cells were fixed, quickly frozen and freeze-fractured to expose the antigen on the fractured surface. They were immunostained with serotonin antibody, and the immunoreactions on the fractured surface were examined on ultrathin sections by electron microscopy. Unstimulated mast cells exhibited serotonin localization mostly in each intragranular matrix. In contrast, mast cells stimulated for 30 s exhibited increased serotonin in their intergranular cytoplasm. Mast cells showed more distinct immunoreactions in the cytoplasm where degranulation would be promoted after 60 s. It is suggested that intracellular release of serotonin occurred in the stimulated mast cells.

Miyagawa, J.; Kuwajima, M.; Hanafusa, T.; Ono, A.; Uenaka, R.; Oue, T.; Yamamoto, K.; Matsuzawa, Y.; Ozaki, K.; Narama, I.; Fujimura, H.; Kaidoh, M.; Nikaido, H.; Hayakawa, J.; Horiuchi, M.; Saheki, T.

Uiker, S.; Kriz, W.

doi: 10.1007/BF00191366pmid: 7773508

The goal of this study has been to characterize the process of glomerular microaneurysm formation and to separate it from capillary ballooning. In the Habu venom model glomerular capillary ballooning and glomerular microaneurysm formation are seen regularly. The sequence of glomerular lesions leading to a glomerular microaneurysm has been examined and it is clear that the process starts with local mesangiolysis. This may proceed to mesangial expansion and/or ballooning of glomerular capillaries but in contrast to ballooning the formation of a glomerular microaneurysm is based on endothelial defects. The process occurs as follows: once initiated by mesangial failure lesions extend along the mesangial axis. As long as the extension of the lesion encroaches on divergent capillary branches, capillary ballooning by “coalescence” is the result. This process comes to an end when convergent capillary branching is reached and two capillaries join. At this point endothelial disruptions occur, blood and mesangial spaces merge and a glomerular microaneurysm is established. Further growth of the microaneurysm occurs following damage spreading along the lobular axis. The entire process has been reconstructed and is presented in a three-dimensional model.