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1.
Gynecol Oncol ; 83(2): 343-7, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11606095

ABSTRACT

OBJECTIVES: The MUC1 antigen can be used to identify epithelial cells from the background of hemopoietic cells. The present investigation describes patterns of overexpression of two novel MUC1 splice variants in human cervical carcinoma cell lines. METHODS: RT-PCR was carried out to determine MUC1 splice variants in the cervical cancer cell lines C-4 II, C-33A, DoTc 2 4510, C-4 I, SiHa, HT3, Hs 636 T (C4-I), and HeLa. RESULTS: The novel MUC1 splice variant D was expressed in all cell lines and the novel MUC1 splice variant C was expressed in all cell lines but C-33A. Variants A and B were expressed in all (variant A) and all but one (variant B) cell line. MUC1/REP was expressed in all cell lines and MUC1/SEC was positive in all but two cell lines (C-33 A, DoTc 2 4510). All but one cell line (C-33A) expressed MUC1/X and MUC1/Y, and two cell lines (C-33 A, DoTc 2 4510) did not express MUC1/Z, respectively. MUC1 variants A, D, and REP could be demonstrated consistently among all eight cervical carcinoma cell lines we have examined. CONCLUSIONS: The present study describes the feasibility of detecting a large number of MUC1 variants, including MUC1 variants C and D which are described for cervical carcinoma cells for the first time. Further studies will examine the presence of MUC1 splice variants' expression in human cervical carcinoma tissue.


Subject(s)
Alternative Splicing , Mucin-1/genetics , Uterine Cervical Neoplasms/genetics , Base Sequence , Female , HeLa Cells , Humans , Molecular Sequence Data , Mucin-1/biosynthesis , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Nucleic Acid , Tumor Cells, Cultured , Uterine Cervical Neoplasms/metabolism
2.
Blood ; 82(1): 262-7, 1993 Jul 01.
Article in English | MEDLINE | ID: mdl-7686787

ABSTRACT

The interaction of human lymphoma cells with high endothelial venules (HEVs) on sections of lymphatic tissues was studied in 44 cases of non-Hodgkin's lymphoma (NHL) with the in vitro HEV binding assay. The relative adherence ratio (RAR) of lymphoma cells to HEVs as related to that of reactive lymphocytes was 0.29 to 4.64 in 38 cases of B chronic lymphocytic leukemia (CLL), 1.15 and 1.54 in two cases of immunocytic NHL, 1.12 and 0.70 in two cases of centrocytic NHL, 1.98 in one case of a peripheral T-NHL, whereas plasma cell leukemia cells adhered very weakly (RAR 0.1). Among the patients suffering from CLL a pronounced HEV binding ability of tumor cells correlated significantly with the more unfavorable Binet stages B and C (median 1.32) as well as with a widespread lymphatic dissemination, which strongly indicates a hematogenous, HEV-mediated spread (median 1.34). In contrast, weak adherence to HEVs was associated with Binet stage A (median 0.85; P < .05) and with a lacking or only localized clinical involvement of lymph nodes (median 0.84; P < .01). Thus, specific HEV recognition processes even operate in lymphoid neoplasms and via this mechanism seem to influence the dissemination of tumors.


Subject(s)
Endothelium, Vascular/cytology , Lymphoma, Non-Hodgkin/pathology , Cell Adhesion , Cell Adhesion Molecules/metabolism , Cells, Cultured , Humans , Immunophenotyping , In Vitro Techniques , L-Selectin , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Lymphoid Tissue/pathology , Neoplasm Metastasis
3.
Lab Invest ; 66(2): 251-60, 1992 Feb.
Article in English | MEDLINE | ID: mdl-1735958

ABSTRACT

The biologic functions mediated by the nuclear protooncogene, c-MYC are correlated to gene dosage. Since automated quantification programs are expensive, time-consuming and not easily available, and since analysis by flow cytometry is difficult in the case of nuclear antigens, we examined the suitability and reproducibility of a semiquantitative in situ evaluation system. This system was based on the percentage of nuclear area staining positively, and comprised the following categories: 0: negative, 1+: single scattered grains of the immunocytochemical staining product, 2+: confluence of grains to patches but less than 50% nuclear area positive, and 3+: greater than 50% positive nuclear area. In addition, sensitivity and specificity of two anti-c-MYC antibodies were investigated. Although both antibodies differed slightly in staining pattern and sensitivity, the four quantification categories were applicable for immunostainings of both antisera and highly reproducible when re-evaluated by the same observer (r = 0.98; p = 0.0001) or a second investigator (rAb155 = 0.98, rAb DCPm = 0.96; p = 0.0001), both reading blindly and independently. Comparing our semiquantitative evaluation categories and results of computer-assisted image analysis, the percentage of positive nuclear area (p less than 0.0001), the median staining intensity (p less than 0.0001), and the product of both (p less than 0.0001) differed significantly in the four evaluation categories. This result still held true after correction for nuclear size, which differed appreciably in various cell types (p less than 0.0001). The product of positive nuclear area, staining intensity and nuclear size (microns 2), which best approximates the absolute amount of c-MYC within a certain cell, was clearly different within the four staining categories (p less than 0.0001) and did not depend on cellular morphology within the staining categories 0 to 2. Also, the immunocytochemical technique proved highly reproducible (median day/day variance 0.65% (0-13); r = 0.995). The practicability of this system for semiquantification was demonstrated by (a) correlation of H score values of immunocytochemical stainings with densitometric scans of Western blots and (b) by the fact that peripheral blood lymphocytes, Phytohemagglutinin stimulated blasts, 13 cases of multiple myeloma and HL-60 cells differed concerning their estimated c-MYC amounts (p = 0.0125). This confirms on the effector molecule level results previously reported from mRNA in situ and Northern blotting analyses. We conclude that a simple and highly reproducible evaluation system can be used for in situ comparison of nuclear oncogene dosage.


Subject(s)
Proto-Oncogene Proteins c-myc/analysis , Gene Expression , Hematopoiesis/genetics , Humans , Image Processing, Computer-Assisted , Immunohistochemistry , Proto-Oncogene Proteins c-myc/genetics , Reproducibility of Results
5.
Blood ; 78(1): 180-91, 1991 Jul 01.
Article in English | MEDLINE | ID: mdl-2070052

ABSTRACT

The c-myc gene plays a pivotal role in mediating the competence state for cell cycle transversion. This biologic role is in contradiction to reports of elevated expression of the gene in multiple myeloma, a tumor with restricted self-renewal capacity. To more clearly define the role of this gene in plasma cells of myeloma patients, c-myc messenger RNA (mRNA) and/or oncoprotein expression were semiquantitatively analyzed on the single cell level in 19 cases of multiple myeloma, among them 1 biclonal case and 1 case with coexistent chronic lymphocytic leukemia (CLL). Performing anti-sense/mRNA in situ hybridization, mature c-myc gene transcripts were detected in 92% (12 of 13) of cases and could definitely be attributed to the plasma cells by our study. The number of Ki 67-positive plasma cells actively passing the cell cycle was less than 1% and independent of c-myc gene expression. However, because the presence of the 152-c-MYC epitope was correlated to extent of marrow plasmacytosis (r = .64; P = .043) and content of plasmablasts (P = .09), the c-myc gene might serve a function different from proliferative activity, but also associated with tumor cell mass. In CLL cells (21 of 22 cases) and their benign counterparts, ie, bone marrow and peripheral blood lymphocytes, the anti-sense/c-myc mRNA hybridization signals remained below the threshold considered as cutpoint between negative and positive. The low amounts of c-myc transcripts were correlated to neither stage of disease (P = .52) nor lymphocyte counts (P = .24). Because the numbers of peripheral blood lymphoma cells were independent of tumor mass and of c-myc gene transcripts expressed, peripheral blood lymphocytosis might more likely reflect homing processes than proliferative activity in CLL.


Subject(s)
Genes, myc/physiology , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Multiple Myeloma/genetics , Antibodies, Monoclonal/immunology , Bone Marrow/metabolism , Bone Marrow/pathology , Cell Differentiation , Cell Division , DNA, Neoplasm/genetics , Gene Expression , Genes, myc/genetics , Humans , Immunohistochemistry , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Multiple Myeloma/pathology , Nucleic Acid Hybridization , Proto-Oncogene Mas , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcription, Genetic
7.
Acta Med Austriaca ; 17(1): 6-10, 1990.
Article in German | MEDLINE | ID: mdl-2191534

ABSTRACT

Proto-oncogenes, the normal equivalents to the transforming genes of mammalian tumorigenic retroviruses, are implicated in essential biological processes of normal human cells, like differentiation and proliferation. In vivo and in vitro studies clearly demonstrate that activation of proto-oncogenes with subsequent transformation in tumorigenic oncogenes plays an important role in induction and acceleration of the malignant disease, and also determines metastatic spread and development of resistance to chemotherapy in certain neoplasias. Investigation of these genes and analysis of their activation state should routinely be introduced in staging of hematological neoplasias, thus contributing to refinement of histopathological classification, clearer discrimination between reactive and neoplastic conditions and understanding of pathophysiological processes. Furthermore, impact on definition of high risk patients and novel therapeutic concepts based on a better definition of tumour biology may be expected from these studies. "Molecular cytology" combines detection of oncogenetic mRNA and the relevant oncoprotein on the single cell level by using "mRNA-in situ hybridization" and immuno-histochemistry. Application of this technique will allow to determine the mechanisms regulating the expression of oncogenes to define functional heterogeneity of tumour cell subsets and to clarify the relevance of minimal residual disease.


Subject(s)
Cell Transformation, Neoplastic/genetics , Gene Expression Regulation, Leukemic/physiology , Gene Expression Regulation, Neoplastic/physiology , Leukemia/genetics , Lymphoma/genetics , Oncogenes/physiology , Proto-Oncogenes/physiology , Humans , Proto-Oncogene Proteins/genetics
8.
Lab Invest ; 60(4): 574-82, 1989 Apr.
Article in English | MEDLINE | ID: mdl-2651801

ABSTRACT

We investigated the practical value of antisense RNA/mRNA in situ hybridization for the detection of low level expression of the c-abl oncogene in non-Hodgkin lymphomas. This is of clinical relevance, since we recently showed that low level expression of this proto-oncogene mainly occurs in advanced stage disease of non-Hodgkin lymphomas and in cases of chronic lymphocytic leukemia with progressive course of the disease (Greil R, Gattringer C, Fasching B, Cleveland J, Thaler J, Radaskiewicz T, Gastl G, Huber C, Rapp U, Huber H: Int J Cancer 42:529 1988). When numerous technical parameters were tested for the adaptation of the method, fixation with 4% paraformaldehyde, gelatin coating of the slides, the time concentration product of proteinase K, and the kind of labeling had the greatest impact on results and successful performance of the technique. When the optimized method was applied to the v-abl-transformed NIH 3T3-, the K 562 CML blast cell line and to nine cases of lowly malignant non-Hodgkin lymphomas it semiquantitatively discriminated the varying amounts of v-abl, bcr/c-abl and c-abl mRNA expressed within these cells. Parallel analysis with Northern blotting confirmed the specificity of the method and pointed to a very high sensitivity, including the capacity to detect only few c-abl mRNA molecules/cell. An essential advantage of in situ hybridization was the detection of inhomogeneous expression of the c-abl mRNA within subpopulations of the malignant clone. In addition, this technique might be of particular importance when a gene is only weakly expressed on a small fraction of cells which might easily escape the detection by Northern blotting. Immunocytochemical investigation suggested parallel expression of the oncoprotein in six of seven c-abl mRNA positive cases as well as high specificity and sensitivity for the polyclonal and to a lesser extent for one monoclonal antibody. However, because of the high potential of cross-reactivity of anti-oncoprotein antibodies, parallel investigations on the mRNA level should be performed particularly when new anti-oncoprotein antibodies are applied. Our results demonstrate that this can be performed using in situ hybridization, even when the number of mRNA targets is very low.


Subject(s)
Antibodies, Neoplasm , Lymphoma/analysis , Molecular Probes , Nucleic Acid Hybridization , Proto-Oncogene Proteins/analysis , Proto-Oncogenes , RNA, Messenger/analysis , Antibodies, Monoclonal , Blotting, Northern , Cell Line , Humans , Immunohistochemistry , Lymphoma/genetics , Lymphoma/pathology , Proto-Oncogene Mas , Proto-Oncogene Proteins/immunology , Proto-Oncogene Proteins c-abl
9.
Int J Cancer ; 42(4): 529-38, 1988 Oct 15.
Article in English | MEDLINE | ID: mdl-3049401

ABSTRACT

Eight reactive lymphatic tissues, 166 cases of non-Hodgkin lymphomas (NHL) and 11 cases of multiple myeloma were investigated for expression of the c-abl protein using the poly-clonal anti-abl antibody 4411 and an indirect peroxidase technique. In selected cases the results were compared to those obtained with a second polyclonal and 2 monoclonal anti-abl antibodies. In 7 cases, Northern blot analysis of abl-mRNA was performed in parallel. In reactive lymphatic tissues, cells positive for the 4411 antibody were confined to the B-cell areas, i.e., to the mantle zone and parts of the germinal center. In NHL, a positive staining of the cell membrane was predominantly detectable in lymphomas putatively originating in the germinal center or mantle zone (in particular in centrocytic NHL), independent of their proliferative activity. Clinically, 7 out of 8 abl-positive cases of chronic lymphocytic leukemia (CLL) had a more aggressive course of disease, whereas "progressive disease" occurred in only 7 out of 19 c-abl antigen-negative cases. When the clinical status of 78 patients with NHL and 11 patients with multiple myeloma was related to c-abl expression, c-abl-positivity was mostly confined to patients in advanced tumor stages [p less than 0.001 (NHL)].


Subject(s)
Lymphoma, Non-Hodgkin/genetics , Proto-Oncogenes , Animals , Cell Differentiation , Humans , Lymphoma, Non-Hodgkin/pathology , Mice , Multiple Myeloma/genetics , Neoplasm Staging , Proto-Oncogene Proteins/analysis , Proto-Oncogene Proteins/immunology , Proto-Oncogene Proteins c-abl , RNA, Messenger/analysis , Rabbits
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