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Biomedical subjects

G Roos

Publications and source records attributed to G Roos.

At least 127 records · Page 7Linked to original sources
PubMed1993

Expression of oncoprotein 18 in human leukemias and lymphomas.

Oncoprotein 18 (Op18) is an 18 kDa intracellular phosphoprotein that appears to be up-regulated in certain neoplastic cells. In the present report we have analysed the expression of Op18 in samples of human hematopoietic disorders, mainly leukemias and lymphomas. For this purpose we have developed reagents allowing quantitative Western-blot analysis, and quantification of Op18 on the single cell level by flow cytometric analysis. The data demonstrates that a significant fraction of all lymphoma and leukemia cases express Op18 at levels that are several-fold higher than the Op18 levels ever found in benign proliferating tissue, such as bone marrows in remission and reactive lymph nodes. Thus, the results establish on the single cell level that Op18 is frequently expressed at abnormal levels in lymphoid and myeloid malignancies. Flow cytometric analysis revealed a striking qualitative and quantitative heterogeneity in Op18 expression between patients with lymphoma or leukemia. Moreover, our analysis demonstrates that the abnormal cellular levels of Op18 expression frequently found in high grade lymphoma and acute leukemia samples do not correlate with an increased fraction of cells in S phase. Finally, we also present an example of dramatic changes in Op18 expression pattern in bone marrow cells during the progression of a chemo-resistant acute leukemia.

Acute Disease↗
PubMed1993

Quantitative analysis of the expression and regulation of an activation-regulated phosphoprotein (oncoprotein 18) in normal and neoplastic cells.

Activation of protein kinase C results in phosphorylation of a 19-kDa protein termed 19K. Isolation and sequence analysis of a cDNA encoding the 19K protein revealed that this protein has been studied in other systems under different names. The name oncoprotein 18 (Op18) has been proposed on the basis of a postulated up-regulation in neoplastic cells. In the present report we adopt the designation Op18 for the 19K protein, and quantify this phosphoprotein in a series of leukemia/lymphoma cell lines, a panel of non-transformed cells and some terminally differentiated cell types. For this purpose we have developed reagents allowing quantitative Western-blot analysis, and quantification of Op18 on the single cell level by flow cytometric analysis. The data demonstrates a pronounced up-regulation of the Op18 protein in most leukemia/lymphoma cell lines. The HPB-ALL cell line provided the most extreme case and expressed 7 x 10(6) Op18 molecules/cell, which compares with 0.65 x 10(6) Op18 molecules/cell in non-transformed lymphoblastoid cells. The expression of Op18 appears to be restricted to cell types with proliferative potential, but it is clear from our results that up-regulation of Op18 is uncoupled from cellular proliferation. Moreover, by employing an Epstein-Barr virus based shuttle vector, we expressed Op18 cDNA in lymphoblastoid cells. This resulted in a three to fourfold up-regulation of Op18 that did not have any detectable consequences for cell-surface phenotype or cell size. However, increased expression of Op18 resulted in a partial inhibition of cell proliferation. Taken altogether, the results suggest that up-regulation Op18 levels in leukemia/lymphoma cells are strongly associated with, but not a direct cause of tumour progression.

Blotting, Western↗
PubMed1992

Breast carcinoma growth rate described by mammographic doubling time and S-phase fraction. Correlations to clinical and histopathologic factors in a screened population.

BACKGROUND: In a retrospective study, correlations among mammographic doubling times (DT), clinicopathologic prognostic factors, and cytometric predictors were examined. METHODS: One hundred fifty-eight patients with the possibility to calculate mammographic tumor DT were selected and the tumors were histologically reexamined and flow cytometric analysis for ploidy and S-phase fraction (SPF) was performed. RESULTS: The tumors were Stage I in 68%, and 45% were detected by mammographic screening. DT ranged from 0.6 months to an indefinite time (median, 9.0 months). Short DT was significantly correlated to large tumor size (P = 0.01) and advanced pathologic tumor stage (P = 0.016), but there was no correlation between DT and histologic grade. Ploidy analysis indicated that there were 57% aneuploid and 7% tetraploid tumors. There was a significant overrepresentation of euploid tumors among tumors smaller than 10 mm (P = 0.02). Ploidy was correlated to histologic grade (P less than 0.001) and DT (P = 0.009). SPF was calculated in 122 cases. SPF correlated significantly with pathologic stage (P = 0.002), tumor size (P = 0.037), histologic grade (P = 0.001), the presence of axillary lymph node metastases (P = 0.046), DT (P = 0.02), and DNA ploidy (P less than 0.001). Compared with interval carcinoma, screening-detected carcinoma showed favorable characteristics concerning size, stage, DT, ploidy, and SPF but not regarding histologic grade and axillary lymph node metastases. CONCLUSIONS: DT shows great variations. Factors related to tumor biology (i.e., DT, DNA ploidy, and SPF) are strongly correlated with one another, but they have no correlation with axillary lymph node metastases. Cancer detected by screening is discovered at an early stage and shows favorable characteristics concerning DT, ploidy, and SPF.

Adult↗
PubMed1992

Mammographic growth rate, DNA ploidy, and S-phase fraction analysis in breast carcinoma. A prognostic evaluation in a screened population.

BACKGROUND: The authors examined prognostic factors in 158 cases of breast carcinoma with known mammographic tumor volume doubling times (DT). METHODS: The tumors were retrospectively reexamined histologically and flow cytometric analysis of DNA ploidy and S-phase fraction (SPF) was performed on archival paraffin-embedded material in each case. Life tables and Cox multivariate analyses were used for statistical evaluation of prognostic factors. RESULTS: In univariate analysis of survival data, clinical and pathologic stage, histologic grade, the presence of axillary lymph node metastases, and SPF were significant prognostic predictors, but mammographic DT and DNA ploidy were not. SPF also contributed prognostic information in the subgroup of carcinoma cases detected by screening. In a Cox multivariate analysis, SPF, the presence of axillary lymph node metastases, and Stage II-III disease (as opposed to Stage I disease) were independent significant predictors of survival. In univariate analyses of distant disease-free survival, clinical and pathologic stage, tumor size, histologic grade, the presence of involved axillary nodes, DT, and SPF all were significant prognostic factors. CONCLUSIONS: SPF, stage, and lymph node status were important prognostic factors in this patient material with predominantly small and node-negative breast carcinomas, whereas DNA ploidy and mammographic DT provided less prognostic information. The prognosis of carcinoma detected during screening did not differ significantly from that of breast carcinoma discovered otherwise in this selected patient group.

Adult↗
PubMed1992

Influence of free thiol group(s) on autoantibody-defined epitope of proliferating cell nuclear antigen.

Proliferating cell nuclear Ag (PCNA) is an intranuclear protein involved in DNA replication directed by DNA polymerase delta and is the target Ag of autoantibodies in some patients with SLE. There is evidence that the epitope on PCNA recognized by human autoantibodies is conformation-dependent and is not a continuous peptide sequence. Thimerosal, a mercury-containing sulfhydryl blocking compound, markedly reduced or abolished the reactivity of this autoantibody-defined PCNA epitope. The thimerosal effect was observed in various Ag-detecting systems including indirect immunofluorescence, immunodiffusion, immunoprecipitation, and flow cytometry. The mechanism of the thimerosal effect appeared to be mediated through free but not readily accessible sulfhydryl group or groups. The sulfhydryl-modification by thimerosal could be reversed by competition with thiol-containing compounds. Experimentally induced mAb to PCNA generated by immunization with purified PCNA have been shown to recognize epitopes that are continuous peptide sequences and these epitopes were not affected by thimerosal. It has been shown that the human autoantibody-defined epitope is related to the function of PCNA because autoantibodies are able to inhibit DNA polymerase delta directed DNA replication, whereas experimentally induced antibodies are not. These studies show that certain sulfhydryl groups in PCNA have a role in determining the antigenicity of the epitope recognized by autoantibody and raise the possibility that certain sulfhydryl groups might also be associated with its function.

Autoantibodies↗
PubMed1992

Flow cytometric analysis of proliferation associated nuclear antigens using washless staining of unfixed cells.

The expression of different proliferation associated nuclear antigens was analyzed using a washless double-staining method and flow cytometry. It is a simple and rapid two-step procedure which can be performed on low cell numbers. A series of hematopoietic cell lines and fresh lymphoma cells were tested and the methodology was found to be applicable to a number of nuclear antigens (PCNA, Ki-67, p105, MPM-2, fibrillarin). For PCNA, the detectability was dependent on the type of antibody used. The immunofluorescence pattern observed by microscopy was altered for antigens stained by the washless technique in comparison with the pattern obtained with fixed cells. With the washless method, detailed cell cycle analysis could be obtained by dual parameter analysis of PCNA and Ki-67.

Antibodies↗
PubMed1992

Colorectal cancer risk after jejunoileal bypass: dysplasia and DNA content in longtime follow-up of patients operated on for morbid obesity.

Jejunoileal bypass (JIB) has been a widespread operation for treatment of morbid obesity. Bile acids are regarded as cofactors in the carcinogenesis of the colon, and, since intestinal bypass involves increased exposure of bile acids to the large intestinal mucosa, JIB has been postulated to increase the risk for colorectal carcinoma. In experimental studies on animals, the results have indicated an increased frequency of induced carcinomas, but in clinical series only one patient with colon carcinoma has been reported. Thirty women, operated on with JIB 11 to 17 years earlier, were examined by colonoscopy with multiple biopsies, systematically taken for histologic evaluation and flow cytometric DNA analysis. In only one patient, low-grade dysplasia was detected in an initial adenomatous lesion but was not visible macroscopically. No DNA aneuploidy was found. In a control group of 11 patients examined for non-neoplastic disease, neither dysplasia nor aneuploidy was diagnosed. Within 17 years postoperatively, we have, by these methods, not been able to verify any colorectal malignant transformation in patients operated on with JIB. However, since carcinogenesis is a long process, further surveillance will be demanded before an increased risk for colorectal carcinoma can be excluded.

Adult↗
PubMed1992

Incorporation of 5-fluorouracil into rat liver tumor and normal tissues and the liver nucleotide profile after administration by the hepatic artery during portal venous branch ligation.

A therapeutic dose of labelled 5-fluorouracil (5-FU) was infused via the hepatic artery during 30 min with or without ligation of the left portal venous branch in Wistar rats with a secondary liver cancer in the left lateral lobe. After another 60 min, the incorporation of 5-FU into the acid soluble fraction (ASF), ribonucleic acid (RNA) and deoxyribonucleic acid (DNA), was determined in tumor, ligated and unligated liver lobes, small intestine, kidney, and bone marrow. The liver nucleotide profile was examined with isotachophoresis. Portal venous branch ligation (PVBL) caused the following changes, compared with the unligated control group: in the tumor, the incorporation of 5-FU into RNA and DNA decreased and the ratio RNA/acid-soluble fraction labelling decreased. The incorporation increased in intestinal and bone marrow RNA. It was unchanged in liver and kidney. The ratio of tumor to peripheral normal-tissue (small intestine, bone marrow, and kidney) labelling of RNA and DNA decreased. Liver nucleotides (F)UTP, (F)UDP-glucuronic acid, (F)UDP-N-acetylhexosamine, and NAD were lower in the ligated than in the unligated liver lobe. ATP and energy charge did not decrease significantly. In conclusion, PVBL in conjunction with hepatic arterial administration of 5-FU increased systemic drug exposure and possibly decreased hepatic tumor anabolism. It has not been examined how this interferes with the therapeutic effect.

Animals↗
PubMed1991

Antibodies to proliferating cell nuclear antigen as S-phase probes in flow cytometric cell cycle analysis.

The usefulness of different anti-proliferating cell nuclear antigen monoclonal antibodies as S-phase probes in flow cytometric analysis was evaluated after various fixation procedures on human cell lines. With a newly developed detergent extraction/fixation method the monoclonal antibody PC10 acted as a selective S-phase marker. A total of 27 human hematopoietic tumors were analyzed using PC10, and all exhibited the characteristic S-phase recognition pattern. The percentage of PC10-positive cells was easily calculated and showed strong correlation to the fraction of S-phase cells determined from DNA histograms. Using alternative fixation procedures PC10, on the other hand, could recognize all actively cycling cells, a feature also observed for the monoclonal antibodies, 19A2 and TOB7.

Antibodies, Monoclonal↗
PubMed1991

Human autoantibody to a novel protein of the nuclear coiled body: immunological characterization and cDNA cloning of p80-coilin.

Antibodies producing an unusual immunofluorescent pattern were identified in the sera of patients with diverse autoimmune features. This pattern was characterized by the presence of up to six round discrete nuclear bodies in interphase cell nuclei. Immunoblotting analysis showed that these sera recognized an 80-kD nuclear protein, and affinity-purified anti-p80 antibody from the protein band reproduced the fluorescent staining of nuclear bodies. Colloidal gold immunoelectron microscopy showed that the affinity-purified anti-p80 antibody recognized the coiled body, an ultramicroscopic nuclear structure probably first described by the Spanish cytologist Ramon y Cajal. Five cDNA clones were isolated from a MOLT-4 cell lambda gt-11 expression library using human antibody and oligonucleotide probes. The longest cDNA insert was 2.1 kb and had an open reading frame of 405 amino acids. A clone encoding a 14-kD COOH-terminal region of the protein was used for expression of a beta-galactosidase fusion protein. An epitope was present in this COOH-terminal 14-kD region, which was recognized by 18 of 20 sera with anti-p80 reactivity, and affinity-purified antibody from the recombinant protein also reacted in immunofluorescence to show specific staining of the coiled body. This is the first demonstration and molecular cloning of a protein that appears to have particular identification with the coiled body, and it was designated p80-coilin. Autoantibody to p80-coilin may be useful for the elucidation of the structure and function of the coiled body, and the availability of a cDNA sequence could be helpful in further studies to clarify the clinical significance of this autoantibody response.

Amino Acid Sequence↗
PubMed1991

Multiple DNA rearrangements in the BCL2 region in a patient with follicular lymphoma.

A 39-year-old male with follicular non-Hodgkin's lymphoma was repeatedly studied with respect to DNA rearrangements with the two probes pFL-1 and pFL-2, representing two segments of chromosome 18. The oncogene BCL2, detected by pFL-1, was as expected translocated to the J region of the immunoglobulin locus. The standard BCL2 translocation was found in three samples, one obtained at diagnosis, one ten months later, and one after 5 years. Another translocation was found with the probe pFL-2 hybridizing with a region located about 20 kb 3' from BCL2. This latter rearrangement was found only in the first biopsy, which was obtained at the time of diagnosis, but not in the two later samples, when the morphology of the lymphoma was unchanged. No cytotoxic therapy had been given in the interval from diagnosis to disappearance of the latter rearrangement. Thus, one of the observed translocations (pFL-2) was detected only in the first biopsy, while the other (pFL-1) was a clonal marker in all three biopsies. The finding suggests that clonal evolution does not necessarily mean clinical progression.

Adult↗
PubMed1991

Immunological and ultrastructural studies of the nuclear coiled body with autoimmune antibodies.

Studies with human autoimmune sera identified auto-antibodies reacting with a novel antigen of 80 kDa. In interphase mammalian cells, the 80-kDa antigen was enriched in nuclear coiled bodies and was used as a marker for this nuclear structure. This antigen was subsequently named p80-coilin. By light and electron microscopic immunocytochemistry, a number of other antigens were also localized to the coiled body, including components of small nuclear ribonucleoproteins which are involved in the processing of nucleolar and extranucleolar RNA. Although the function of the coiled body is unknown, the presence of these subcellular particles might indicate an involvement in RNA metabolism. The identification of a protein highly enriched in this structure and the availability of specific antibodies might help in its isolation and the study of its function.

Animals↗
PubMed1991

Flow cytometric deoxyribonucleic acid analysis in stage I renal cell carcinoma.

Tumor deoxyribonucleic acid (DNA) content was analyzed by flow cytometry in 60 consecutive patients with stage I renal cell carcinoma. Of 59 evaluable tumors 27 (46%) were homogeneously diploid, 1 (2%) was tetraploid and 31 (52%) were aneuploid. Of the 32 nondiploid tumors 25 were heterogeneous concerning ploidy. One of the 27 patients with diploid tumors had metastases compared to 5 of the 32 patients with nondiploid tumors (not significant). There was a significant difference in survival between patients with diploid and nondiploid tumors (p = 0.043). Neither nuclear grade, tumor cell type nor tumor size correlated with survival. Analysis of DNA content seems to predict survival significantly for patients with stage I renal cell carcinoma.

Adult↗
PubMed1991

Reduction by norepinephrine of the side effects induced by combined hepatic arterial administration of degradable strach microspheres and adriamycin in rats with a liver adenocarcinoma.

In previous studies we found degradable starch microspheres (DSM) to increase the antitumor effect of adriamycin injected by the hepatic artery in rats with a liver adenocarcinoma. Increased side effects also appeared, namely body weight loss and liver necroses. In the present paper, norepinephrine in four different protocols was added to the injection of adriamycin + DSM to decrease the drug flow to normal tissues. In two protocols norepinephrine decreased the body weight loss. There was also a non-significant decrease in liver necroses but also in antitumor effect. In these experiments we also observed that some rats given adriamycin + DSM got gastric necroses. This was not found when norepinephrine was added. Addition of propranolol to norepinephrine did not decrease side effects. Vasoactive drugs may therefore be of value to diminish adverse side effects of the combination cytostatic agent + DSM, probably decreasing overspill into normal tissues.

Adenocarcinoma↗
PubMed1991

Cytogenetic and flow cytometric DNA analysis in renal cell carcinoma.

Cytogenetic and flow cytometric DNA analyses were performed on 37 tumor samples from 16 patients with renal cell carcinoma. 36 cultures were chromosomally abnormal and 1 was normal. The most frequent abnormality was trisomy or tetrasomy of chromosome 7 (13 of 16 tumors), suggesting that this abnormality might be a primary change in renal cell carcinoma. Abnormalities of chromosome 3 were seen in 9 of 16 tumors (56%). Eight of 11 tumors (73%) with cytogenetic results from multiple tumor samples showed intratumor cytogenetic heterogeneity. Twelve of the 16 patients had aneuploid tumors, and 4 had homogeneously diploid tumors. DNA index correlated with chromosome number. No association was found between cytogenetic results and clinical stage.

Aneuploidy↗
PubMed1991

Expression of proliferating cell nuclear antigen (PCNA) and Ki-67 antigen in human malignant hematopoietic cells.

Human hematopoietic cell lines and fresh lymphoma cells were investigated for their expression of proliferating cell nuclear antigen (PCNA) and Ki-67 antigen using flow cytometry. Ki-67 was detected with a monoclonal antibody and the PCNA content was determined using a human auto-antiserum and in some cases a monoclonal antibody (PC10). The autoantibody to PCNA was found to recognize S-phase cells and the number of positively stained cells was significantly correlated with the S-phase fraction determined from DNA-histograms. There was also a strong association between Ki-67 positivity and the fraction of S-phase cells. By dual parameter analysis of Ki-67 and PCNA detected by the autoantibody a detailed cell cycle analysis could be performed of the lymphoma samples. The recently described PC10 monoclonal antibody was found by FCM to be a good proliferation marker recognizing cycling cells after methanol and detergent treatment, however without any specific cell cycle phase preference.

Cell Nucleus↗