Joop ten Kate

Transfected c-Ha-ras oncogene enhances karyotypic instability and integrates predominantly in aberrant chromosomes

A human colon tumor cell line, SW480, was transfected with the c-Ha-ras oncogene, the wild type c-Ha-ras gene, or the pSV2neo plasmid. Cytogenetic analysis and localization of chromosome integration sites were combined in an attempt to analyze the effects of transfection with the c-Ha-ras oncogene on the karyotype. All transfected cell lines showed new clonal chromosome abnormalities present in all cells, ranging from three new aberrations in pSV2neo-transfected SW480 cell lines to eight in c-Ha-ras oncogene-transfected SW480 cell lines. The level of expression of c-Ha-ras mRNA after transfection with the c-Ha-ras oncogene was positively correlated with increased genetic instability, reflected in enhanced karyotypic instability. A combination of banding and fluorescence in situ hybridization (FISH) was used to identify chromosome integration sites. Plasmids containing ras integrated predominantly in new structurally rearranged chromosomes (five of eight). Three of five integration sites in new structurally rearranged chromosomes were localized at or near translocation breakpoints situated in telomeric regions. Specific chromosomes were not involved in the chromosome rearrangements. The results indicate that 1) enhanced expression of c-Ha-ras mRNA correlates with an increase in genetic instability in c-Ha-ras oncogene-transfected SW480 cell lines, and 2) that no specific integration site was observed but ras-containing plasmids were located predominantly in aberrant chromosomes near or at translocation breakpoints involving telomeric bands.

Southern and dot blot analysis of DNA from formalin-fixed, paraffin-embedded tissue samples from colonic carcinomas

SummaryTwo extraction methods for the isolation of DNA from formalin-fixed, paraffin-embedded tissue samples from colonic carcinomas were compared. The processed DNAs were compared with DNAs from fresh specimens of the same tumors. The two extraction methods gave similar results. Formalin-fixation and paraffinembedding irreversibly denatured DNA and consequently decreased the extraction yield and interfered with the quantitative measurement of DNA.Southern blot and dot blot analysis of processed and native DNA was performed using a c-myc and an actin probe. The results show that for Southern analysis processed DNA can be used but, due to the generation of random breaks, the restriction fragments have to be small. Furthermore, the fixation-induced crosslinking of DNA appears to hamper hybridization. For these reasons processed DNA can be analyzed better by dot blot rather than Southern blot hybridization.

Immunohistochemical localization of adenosine deaminase complexing protein in intestinal mucosa and in colorectal adenocarcinoma as a marker for tumour cell heterogeneity

SummaryAdenosine deaminase complexing protein (ADCP), a dimeric glycoprotein, has been reported to be decreased or deficient in transformed or cancer-derived cell lines, indicating its potential significance as an indicator of malignant transformation. A similar deficiency was reported in total homogenates of tumours of colon, kidney, lung and liver. In previous biochemical studies we failed to confirm the consistent reduction in ADCP concentration in cancer tissues. A possible explanation for our findings was thought to be intercellular heterogeneity in ADCP expression in individual tumour cells. To study ADCP expression in individual cells we developed an immunohistochemical method which was applied to tissue sections. Paraformaldehyde-lysine-periodate (PLP) solution was found to be a suitable fixative. Fixed tissue samples were paraffin-embedded, sectioned and stained for ADCP, using an indirect peroxidase-labelled antibody procedure. The protein was localized in normal colonic mucosa, mainly in the brush border region of the luminal epithelium and in cytoplasmic granules. Intense ADCP immunoreactivity was found also in the basal part of some cells. In cancer cells, three staining patterns were observed: membranous, diffuse cytoplasmic and granular cytoplasmic. The adenocarcinomas exhibited significant intratumour and intertumour heterogeneity in their staining types.Further studies on ADCP expression in colorectal cancer in relation to clinical and histopathological characteristics are warranted in order to fully evaluate the potential significance of ADCP as a cancer associated antigen.

Cell Surface Adenosine Deaminase (ADA) and its Complexing Protein (ADCP) in Human T-Lymphoid Cells

Adenosine deaminase (ADA, EC no. 3.5.4.4), the enzyme which converts adenosine and deoxyadenosine to inosine and deoxyinosine respectively, plays an important role in the development of the immune system. The enzyme is probably involved in T-lymphocyte differentiation since a deficiency of the enzyme is associated with severe combined immunodeficiency disease.1

Solid-phase adsorption of antigens for efficient production of antibodies reactive with native and fixed tissue antigens☆

A study has been made of the efficacy of different immunization protocols using low antigen levels for the generation of monoclonal antibodies capable of detecting antigens (ADCP) in processed tissues. Protocols using unprocessed native antigen immobilized on nitrocellulose were more efficient than soluble antigen in generating serum antibodies reactive with both native antigen and processed tissues. The derived monoclonal antibodies reacted with native but not processed antigen. The use of antigen immobilized on polyvinylidene (PVDF) and subsequently processed as for histochemistry was successful in generating monoclonal antibodies reactive with processed antigen.

Colonic mucosal changes in nude mice associated with orthotopic xenografts of human colon cancer cells.

We used the nude mouse tumour xenograft model to study the pathogenesis of mucosa alterations in the large bowel surrounding a carcinoma. In mouse colonic mucosa overlying HT-29 colonic carcinoma xenografts in the caecum, the crypts were elongated in comparison with those in distant mucosa and also frequently showed a shift towards sialomucin production. These features, which are comparable with socalled transitional mucosa (TM) in man, were absent in control animals inoculated with Indian Ink instead of HT-29 cells. Although the localization of the proliferative cell compartment in mouse colonic mucosa overlying HT-29 xenografts appeared to be confined to the lower half of the crypt as in normal mucosa, the relative length of the DNA synthesizing cell compartment along the crypts was slightly elongated. These data strongly suggest that TM should be regarded as a secondary phenomenon rather than a premalignant change in large intestinal epithelium and that higher proliferative activity of epithelial cells contributes little to the elongation of crypts in TM.

Adenosine Deaminase (ADA; E.C. no. 3.5.4.4.) in Colorectal Adenocarcinoma in Man

Soluble ADA is known to occur in different isozymes in man. Two major forms have been identified in the normal human tissues: the so called red cell ADA or ADA-S, a monomeric protein, with a M.W. of about 35KD (kilodaltons), and a tissue specific form, or ADA-L, of about 300KD, composed of a dimeric glycoprotein molecule (adenosine deaminase complexing protein=ADCP) and two molecules ADA-S. Additional ADA isozymes have been reported by several authors: a form of 435KD in the normal intestine, a so called particulate form in different tissues probably due to membrane bound ADA4 and a cancer specific 70KD in some colorectal tumors5. Nishihara et al.6 observed that free ADCP, usually present in normal lung tissue, was strongly decreased in the extracts of cancerous lung. Trotta and Balis presented evidence for the absence of ADCP in a clear cell carcinoma of the kidney, in spite of the fact that normal kidney is the major source of free ADCP. In the same report they described a striking decrease of ADA-L, thus of ADCP, in 14 out of 15 colorectal tumors. These data and the data presented by Herbschleb-Voogt et al. (this conference), where ADA-L was found to be almost absent or markedly decreased in 6 transformed- and tumor derived cell lines, formed the basis for the present investigation.

Comparison of DNA Isolation Kits to Extract DNA from Whole Blood Samples

Background and aim Genomic deoxyribonucleic acid (DNA) from white blood cells (WBCs) is widely used for polymerase chain reaction (PCR). Although kits for DNA isolation are in common use, there is scarce information about their performance and the PCR quality of the genomic DNA obtained. Hence, three different kits, QIAamp blood mini kit, High Pure PCR Template Preparation Kit and the Puregene DNA isolation kit were evaluated on these aspects. Materials and methods Genomic DNA was isolated from whole blood samples with WBC counts ranging from 0.5 to 20 x 10 WBC/l. The WBC count was used to calculate the amount of genomic DNA. The actual amount of genomic DNA isolated, was determined spectrophotometrically. The DNA extraction efficiency was obtained by dividing the actual amount of DNA by the calculated amount yielded. PCR quality was analysed by measuring Cycle threshold (Ct) values with a quantitative real-time PCR β-globin assay. Results The extraction efficiency of the three DNA isolation kits was 20% to 40%. Spectrophotometrically determined DNA concentrations correlated inversely with Ct values, regardless of the DNA isolation kit applied, whereas the strongest correlation was obtained for the Puregene DNA isolation kit. WBC counts also correlated inversely with Ct values and here the strongest correlation was found for the QIAamp blood mini kit. Conclusion The overall performance of the DNA isolation kits was quite comparable (DNA recoveries of 20 40%), albeit the QIAamp blood mini kit yielded the most reproducible extraction efficiencies and lowest Ct values within every WBC count category. Comparison of DNA isolation kits⏐59

Maturation Dependent Changes in Nucleoside Phosphorylase (NP), Adenosine Deaminase (ADA) and Ada Complexing Protein (ADCP) in Intestinal Epithelial Cells

Adenosine deaminase (ADA, E.C. no. 3.5.4.4) and purine nucleoside phosphorylase (NP, E.C. no. 2.4.2.1) are two successive enzymes in purine metabolism. A deficiency of the either is known to cause immune deficiency (Giblett et al. 1972, 1975; Stoop et al. 1977), and the ratio of both activities (i.e. ADA/NP) was reported to be inversely related to the degree of maturation of thymocytes (Demeocq et al. 1982, Schuurman et al. 1983). The so called ADA-L, consisting of both ADA and its complexing protein, ADCP, is known to exhibit transformation and cancer associated changes in its expression (Nishihara et al. 1973; Trotta and Balis 1978; ten Kate et al. 1983, 1984a, 1984b; Herbschleb-Voogt et al. 1983). The changes mainly involve the ADCP molecule. While quantitative and immunohistochemical studies on the expression of ADCP in cancer are in progress (ten Kate et al. 1983, 1984a, 1984b) we studied the distribution of ADA and ADCP in the normal intestinal mucosa. Therefore, we have set up a technique to obtain monodispersed epithelial cell preparations. This technique yielded cell fractions with differences in their maturation stages, as deduced from sequential tissue sections studied by light microscopy.

IMMUNOHISTOCHEMICAL DEMONSTRATION OF ADA AND ADCP ON THE CELL SURFACE OF HUMAN T-LYMPHOID CELLS: 54

ADA plays an important role in the development of the immune system and the enzyme is probably associated with T-lymphocyte differentiation. ADA has been shown to exist in different molecular weight forms. In man the major forms are the red cell ADA or ADA-S and the tissue specific ADA or ADA-L. ADA-S can be converted to ADA-L by complexing with ADCP. Intact human peripheral T lymphocytes and human thymocytes were first incubated with rabbit anti-ADA and anti-ADCP antisera, followed by incubation with FITC-labeled swine anti-rabbit IgG. Since only cell surface ADA and ADCP would be exposed to the antisera, fluorescence indicates ADA and ADCP expression on the outer cell membrane.Labeled cells were analyzed by fluorescence microscopy and FACS analysis. ADCP is present at the cell membrane of about 50% of the T lymphocytes; 10-20% of the cells contain membrane-bound ADA. When these cells were incubated with a lysate of human erythrocytes (as source of ADA-S) about 50% of the cells were found to display ADA at the membrane. Human thymocyte suspensions contained 20-30% membrane-ADA positive cells and 10-20% membrane-ADCP positive cells. The physiological significance of ADA and ADCP expression at the extracellular surface of human T-lymphoid cells will be resolved.