Biomedical subjects

G Roos

Publications and source records attributed to G Roos.

At least 73 records · Page 4Linked to original sources

PubMed1998

Loss of heterozygosity at chromosome 3p correlates with telomerase activity in renal cell carcinoma.

Acquired loss of the entire or parts of the short arm of chromosome 3 is a frequent aberration in renal cell carcinoma as well as in other tumour types, indicating the presence of at least one tumour suppressor gene on 3p. Previous studies have defined the distal and proximal ends of one critical region to reside between 3p21 and 3p11, and one gene involved in von Hippel-Lindau disease has been identified at 3p25. Experimental in vitro data has suggested a negative regulator of telomerase activity on chromosome 3. In the present study we investigated the relationship between telomerase activity and loss of heterozygosity (LOH) on 3p in a series of renal cell carcinomas. Telomerase activity was evaluated using the telomeric repeat amplification protocol assay and LOH, by analysis of 17 polymorphic microsatellite markers. Twenty-nine out of 45 tumours (64%) demonstrated telomerase activity and 37 tumours (82%) showed allelic loss of single or multiple areas of chromosome 3p. A significant correlation between LOH of at least one of three markers localised within 4 cM in the region of 3p21.2-3p14.2 and telomerase activity was demonstrated (p=0.0031), as well as for three distal markers within 3 cM at 3p24.3-3p24.1 (p=0.0287). These data suggest the presence of at least two genes with regulatory function on the expression of telomerase. These genes can encode proteins of importance for senescence and/or immortalisation or have a more direct effect on activation of telomerase.

Carcinoma, Renal Cell↗

PubMed1997

Telomerase activity is associated with cell cycle deregulation in human breast cancer.

Deregulation of the cell cycle by abnormal expression of one or several cell cycle regulatory proteins is a common finding in malignant tumors and might be a prerequisite for cancer development. Telomerase activity is an immortalization marker that is found in most cancers and for which an association with an active cell cycle has been implicated. In the tissue of 106 human breast carcinomas, we analyzed the relationship between telomerase activity levels and defects in the cell cycle machinery with a focus on the retinoblastoma protein (pRB) pathway(s). The fraction of telomerase-positive tumors was 85%, and large differences in telomerase activity were found. Overexpression of cyclin D1 and/or cyclin E, in combination with a normal pRB, was a typical feature of tumors with high telomerase activity levels. Down-regulation of p16INK4 was not related per se to telomerase activity, but tumors with low p16INK4 in combination with cyclin D1 or E overexpression demonstrated high activity. Tumor cell proliferation, determined by Ki-67 expression, correlated significantly to telomerase activity levels. There was, however, not a strict association between proliferation rate and telomerase activity, because tumors with inactivated pRB had the highest Ki-67 fractions but intermediate telomerase activity. Also, cyclin D1 overexpression was associated with high telomerase levels without an increase in tumor cell proliferation. The present study indicates that telomerase activation occurs preferentially in breast cancers with certain cell cycle regulatory defects and that telomerase activity levels may depend on the specific defect(s).

Breast Neoplasms↗

PubMed1997

Incorporation of 5-fluorouracil into hepatoma and normal tissue RNA at protein depletion in the rat.

BACKGROUND AND OBJECTIVES: 5-fluorouracil (5-FU) exerts its effects mainly by its incorporation into RNA and inhibition of DNA synthesis. Its toxicity may therefore be estimated by measuring its incorporation into RNA. Protein malnutrition has been considered to increase the toxicity of 5-FU. METHODS: Rats with a hepatoma implanted into the liver were fed on either a 25% or a 0% casein diet for 1 week. On the last day, they were infused via the hepatic artery with a therapeutic dose of 3H-5-FU. Its incorporation into RNA was measured in hepatoma and several normal tissues. RESULTS: Protein deprivation increased the incorporation of 5-FU into liver and intestinal RNA. Incorporation into hepatoma RNA did not increase significantly, but the ratio, liver/hepatoma RNA incorporation, remained unchanged. CONCLUSIONS: Protein deprivation might increase the toxicity of 5-FU on liver and intestine.

Animals↗

PubMed1997

The Heidelberg classification of renal cell tumours.

This paper presents the conclusions of a workshop entitled 'Impact of Molecular Genetics on the Classification of Renal Cell Tumours', which was held in Heidelberg in October 1996. The focus on 'renal cell tumours' excludes any discussion of Wilms' tumour and its variants, or of tumours metastatic to the kidneys. The proposed classification subdivides renal cell tumours into benign and malignant parenchymal neoplasms and, where possible, limits each subcategory to the most commonly documented genetic abnormalities. Benign tumours are subclassified into metanephric adenoma and adenofibroma, papillary renal cell adenoma, and renal oncocytoma. Malignant tumours are subclassified into common or conventional renal cell carcinoma; papillary renal cell carcinoma; chromophobe renal cell carcinoma; collecting duct carcinoma, with medullary carcinoma of the kidney; and renal cell carcinoma, unclassified. This classification is based on current genetic knowledge, correlates with recognizable histological findings, and is applicable to routine diagnostic practice.

Humans↗

PubMed1997

Immunoglobulin VH gene replacements in a T-cell lymphoblastic lymphoma.

We have analysed the rearrangement status of the immunoglobulin heavy (IgH) chain locus during progression of a T-cell lymphoblastic lymphoma displaying multiple IgH rearrangements as demonstrated by variable heavy (VH) gene family specific polymerase chain reaction (PCR) analysis. The tumor was found to undergo diversification at the IgH locus between diagnosis and relapse through a mechanism of VH to VHDJH replacement. In subsets of the tumor at relapse, two separate VH gene segments were found to have replaced the VH gene utilized by a VHDJH rearrangement identified at diagnosis. The observed VH gene replacement events appear to have been mediated by a heptamer sequence homologous to the heptamer of the recombination signal sequence (RSS) located internally in the VH gene segment. These results support the notion that VH replacements contribute to the diversification of immunoglobulin genes.

Adult↗

PubMed1997

Linell classification of breast cancer morphology compared to histologic grading, S-phase fraction and DNA-ploidy.

One hundred and fifty-eight histologically verified mammary carcinomas with known mammographic doubling time (DT) were studied with special emphasis on a morphologic classification proposed by Linell et al. [8, 12, 14, 15]. The hypothesis that Linell classification of ductal carcinomas into comedo, tubuloductal and tubular carcinomas is easy to perform with small inter-observer variations, was not fully confirmed. The Linell classification was found to correlate well with conventional WHO malignancy grading, S-phase fraction and DNA-ploidy. The Linell classification also correlated to surgical stage, lymph node status and DT, but not at all to tumour size. Using distant disease-free survival as an endpoint, the Linell classification gave prognostic information comparable to conventional histologic grading, seeming to be a simple, cheap and reliable method well worth trying on a larger scale.

Adenocarcinoma↗

PubMed1997

Telomeres and telomerase in normal and malignant haematopoietic cells.

The normal haematopoietic system harbours telomerase-competent cells with a capacity to upregulate the activity to notable levels in a telomere length-independent manner. Strong telomerase activity is found in progenitor stem cells and activated lymphocytes in vitro as well as in vivo, indicating that cells with high growth requirements can readily upregulate telomerase. Despite detection of telomerase activity, a gradual telomere erosion occurs in stem cells and lymphocytes, with significantly shortened telomeres at higher ages, a phenomenon that might be of importance for developing immunosenescence and exhausted haematopoiesis. In malignant haematopoietic disorders telomerase activity is a general finding with large differences in activity levels. The strongest telomerase expression has been shown in acute leukaemias and non-Hodgkin's lymphomas, especially high grade cases. There are indications that the level of activity might parallel tumour progression and be of prognostic relevance, but studies of larger patient materials are needed. An association between the cell cycle and telomerase activity exists, especially for normal haematopoietic cells, and induction of a differentiation programme in immortalised cell lines downregulates telomerase activity. The expression of telomerase activity seems to be regulated at different levels, since for immature bone marrow cells the level of activity seemed to parallel better the phenotype than the proliferation state. The frequent expression of telomerase in leukaemias and lymphomas makes these disorders interesting targets for future anti-telomerase therapy.

Hematopoietic Stem Cells↗

PubMed1997

Novel variants of human IFN-alpha detected in tumor cell lines and biopsy specimens.

Interferon-alpha constitutes a complex gene family with 14 genes clustered on the short arm of chromosome 9. More than 50 sequence variants have been described. However, an extensive genetic polymorphism has not been seen in the few population studies reported so far. As many of the sequence variants reported were derived from tumor cell lines, we have investigated whether IFN-alpha genes are unstable in tumor cells. Using fluorescence-assisted mismatch analysis (FAMA), combined with allele-specific primer extension, RFLP analysis, and direct sequencing, we detected in a panel of 14 tumor cell lines two new sequence variants of the IFNA1 and IFNA13 genes. Further two-point mutations were found in tumor samples from leukemias (n = 10) and renal cell carcinomas (n = 17) not seen in normal tissues. In the IFNA17 gene, three new sequence variants were detected, one in a tumor cell line and two in tumor biopsy specimens. Besides these individual point mutations, two new polymorphisms were found in each of the IFNA13 and IFNA17 genes. No new variants were found in the IFNA2 and IFNA10 genes. The results suggest that new sequence variants of the IFN-alpha genes occur relatively frequently in tumors or in tumor cell lines.

Biopsy↗

PubMed1997

Comparative analysis of detection systems for evaluation of PCR amplified immunoglobulin heavy-chain gene rearrangements.

Four different detection systems were compared for evaluation of polymerase chain reaction (PCR) amplified immunoglobulin heavy-chain gene rearrangements in acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma (NHL) of B-cell lineage. In 63.0% of the fragments detected by ethidium bromide stained agarose gel electrophoresis (Agarose-EtBr) the sensitivity was insufficient to separate the specific clonal population from the background of normal B cells. Using polyacrylamide gel electrophoresis (PAGE), PAGE combined with single-strand conformation polymorphism (PAGE-SSCP) and PhastGel-SSCP (Phast-SSCP) analysis with silver staining, the resolution was improved and the majority of the inconclusive amplicons were elucidated. However, Phast-SSCP displayed a slightly higher detection level compared to PAGE and PAGE-SSCP. According to our findings PAGE-SSCP and Phast-SSCP were superior to agarose-EtBr and PAGE in detecting new emerging clones and clonal evolution.

DNA, Neoplasm↗

PubMed1997

The cell cycle in breast cancer.

Breast cancer is a heterogeneous disease regarding morphology, invasive behavior, metastatic capacity, hormone receptor expression and clinical outcome. For prediction of prognosis, tumor cell kinetics is an important feature, traditionally evaluated by estimation of cell growth-associated parameters such as mitotic index, S-phase fraction and expression of proliferation coupled proteins, for example proliferating cell nuclear antigen (PCNA) and Ki-67 antigen. Recent data indicate that deregulation of the cell cycle can occur at different levels in cancer and that the "deregulation pattern" can be of clinical significance. In the present overview we give a short description of approaches used for cell proliferation assessments, whereafter more recent data on cell cycle deregulation are discussed. Alterations of importance in breast cancer include overexpression of cyclins D1 and E, down-regulation of cyclin-dependent kinase inhibitors, such as p16, and inactivation of the retinoblastoma and p53 tumor suppressor proteins.

Biomarkers, Tumor↗

PubMed1997

PCNA, Ki-67, p53, bcl-2 and prognosis in intraoral squamous cell carcinoma of the head and neck.

Eighty patients with primary intraoral squamous cell carcinomas of the head and neck, with a follow-up of 4-14 years were analysed for clinical outcome in relation to immunohistochemical expression of PCNA, Ki-67, p53, bcl-2 and presence of mutations in the p53 gene. The tumor site was not associated with the different parameters calculated. PCNA and Ki-67 labelling showed median values of 56% and 32%, respectively, and neither antigen was of predictive value. Fifty-five percent of the tumours expressed p53, and 38 (48%) had mutations in the p53 gene. No association between the presence of p53 protein or mutations in the p53 gene and clinical outcome was found. Bcl-2 positivity was detected in a minor fraction (10%) of the tumours.

Adult↗

PubMed1997

Flow cytometric evaluation of proliferating cell nuclear antigen expression in human hematopoietic malignancies.

Cell proliferation is a strong prognostic factor in various malignancies including non-Hodgkin's lymphoma's (NHL). Several methods to evaluate tumour proliferation are available based on immunohistochemical and flow cytometric techniques, but none has been widely accepted for multicenter studies. In the present study 51 samples from patients with haematological disorders were analysed for the expression of proliferating cell nuclear antigen (PCNA) by a previously described flow cytometric approach. S-phase specific PCNA (PCNA-S) as well as growth fraction-associated PCNA (PCNA-tot) expression were evaluated. The mean value for PCNA-S was 9.0% and for PCNA-tot 17,4%. PCNA-S and PCNA-tot correlated strongly to each other (r(s) = 0.969, p < 0.001) and to the S-phase fraction determined by DNA histogram analysis (r(s) = 0.927 and 0.934 respectively, p < 0.001). In 23 cases with NHL in vivo iododeoxyuridine (IdUrd) labelling was performed to assess the labelling index (IdUrd-LI, i.e. S-phase fraction), S-phase duration time (Ts) and potential tumour doubling time (Tpot). IdUrd-Li correlated significantly to both PCNA-S and PCNA-tot (r(s) = 0.704 and 0.622 respectively, p < 0.001 and 0.02). In conclusion, especially the PCNA-S seemed to be a candidate for future larger studies of proliferation related aspects of haematological malignancies.

Affinity Labels↗

PubMed1996

Nuclear autoantigen p330d/CENP-F: a marker for cell proliferation in human malignancies.

p330d, also known as CENP-F, is a newly characterized cell cycle specific nuclear autoantigen which is associated both with the centromeres and the nuclear matrix. It is expressed in low amounts in G0/G1-cells and accumulates in the nuclear matrix during S-phase with a maximum expression in G2/M-cells. In the present study we have investigated if p330d/CENP-F could be used as a marker for proliferation in different human malignancies. A flow cytometric method was developed by which p330d/CENP-F expression and DNA-content could be assessed on hematopoietic and solid tumors. Twenty-four different human hematopoietic malignancies, 12 breast cancers, and several cell lines were analyzed and the number of p330d/CENP-F positive cells and the S-phase fraction were determined. The percentage of p330d/CENP-F positive cells correlated with the fraction of S-phase cells in all human malignancies tested. Various cell lines revealed a similar cell cycle specific distribution. The association of p330d/CENP-F with the nuclear matrix facilitated the flow cytometric analysis of this protein due to its resistance to different preparation and fixation procedures. In summary, p330d/CENP-F seems to be a potentially valuable proliferation marker which can be applied to different tumors.

Antigens, Neoplasm↗

PubMed1996

Determination of the epitope of an inhibitory antibody to proliferating cell nuclear antigen.

Proliferating cell nuclear antigen (PCNA) is an essential component for the normal processive DNA synthesis by DNA polymerase delta and is also required for DNA excision repair. Human PCNA autoantisera has been shown to inhibit the function of PCNA in vitro, in contrast to induced antibodies. A monoclonal IgG2 PCNA antibody, 74B1, that effectively inhibited DNA replication in vitro was identified. The inhibitory effect was dose dependent and using synthetic overlapping peptides of PCNA the 74B1 epitope was mapped to aa 121-135, a region of the PCNA protein containing the interdomain connector implicated in intermolecular interactions. Interestingly, a neighboring and partly overlapping peptide, aa 111-125, contains an immunodominant region recognized by a number of monoclonal PCNA antibodies with no inhibitory effect on DNA synthesis. The 74B1 antibody is a potentially useful antibody in future studies of PCNA and its interaction in complexes associated with cell cycle progress, DNA replication, and DNA repair.

Amino Acid Sequence↗

PubMed1996

Telomerase activity in human renal cell carcinoma.

Telomeres have a vital role in maintaining chromosome stability and are essential for long term viability. Since the very ends of linear chromosomes cannot replicate, telomeres shorten in normal somatic cells eventually resulting in growth inhibition. However, most immortal cell lines maintain stable telomeres indicating that mechanisms exist to compensate for the end replication problem. Telomerase activity, leading to synthesis of telomeric DNA repeats, has been proposed to be an important step in the immortalization process of tumor cells. In the present study, 56 renal cell carcinomas were tested for telomerase activity using the sensitive TRAP (telomeric repeat amplification protocol). Forty of the analysed tumors (71%) were positive for telomerase activity, whereas none of the 56 corresponding normal kidney samples showed telomerase activity. All telomerase negative tumors had a reduction in mean telomere restriction fragment (TRF) length and a decrease in total telomere repeat hybridization signal, though cases were observed with an increase in peak TRF lengths. No obvious association between the presence of telomerase activity and clinicopathological parameters (histopathologic grade, DNA-ploidy, stage and clinical outcome) was found. The high frequency of detection of telomerase activity in the renal cell carcinomas indicates that this enzyme is likely to be an important factor involved in the evolution of this tumor type.

Base Sequence↗

PubMed1996

Telomerase activation in normal B lymphocytes and non-Hodgkin's lymphomas.

Activation of telomerase seems to be a prerequisite for immortalization and is found in permanent cell lines and most malignant tumors. Normal somatic cells are generally telomerase negative, except for bone marrow stem cells. Weak activity is also present in peripheral blood cells. In the present study strong telomerase activity was demonstrated in vivo in normal mature cells of the immune system, as well as in malignant lymphomas. Benign lymph nodes had lower telomerase activity than benign tonsils, which exhibited intermediate to high activity comparable with findings in malignant lymphomas. In benign tonsils the activity seemed to be restricted to germinal center B cells. In benign lymphoid tissues telomerase activity correlated with B-cell numbers and cell proliferation, but this was not observed in the lymphoma group. High-grade lymphomas exhibited higher levels of telomerase compared with low-grade cases. The data showed that in vivo activation of telomerase is a characteristic feature of germinal center B cells. Different signals for activation of telomerase are likely to exist, one of them being immune stimulation. The data suggest that telomerase activity in malignant lymphomas can be explained by an "induction and retention" model, ie, transformation occurs in a normal, mature B cell with reactivated telomerase, which is retained in the neoplastic clone.

B-Lymphocytes↗

PubMed1996

Cell proliferation in renal cell carcinoma--a comparative study of cell kinetic methods.

Four different methods of assessing cell proliferation in renal cell carcinoma were compared in a total of 136 samples to analyze their degree of agreement and usefulness. The methods compared were flow cytometric S-phase (S-FCM) analysis, proliferating cell nuclear antigen expression detected by immunohistochemistry (PCNA-IHC), in vivo iododeoxyuridine incorporation analyzed with immunohistochemistry (IdUrd-IHC), and flow cytometry (IdUrd-FCM). The mean S-FCM fraction was 5.9%, compared with a mean PCNA-IHC labeling index of 4.7%. The mean labeling indices obtained by IdUrd-IHC and IdUrd-FCM were 1.2% and 1.7%, respectively. The four methods correlated well with each other. When the methods were compared according to Bland and Altman, good agreement was shown. A statistically significant difference in proliferation between diploid and aneuploid tumor samples was found with all methods (P < 0.001). The results showed that the four different methods provided comparable information on proliferative activity, although different cell cycle compartments were monitored.

Aneuploidy↗

PubMed1996

A non-random deletion in the p53 gene in oral squamous cell carcinoma.

In a retrospective study of the mutational spectrum of the p53 gene in oral squamous cell carcinoma, 80 primary tumours diagnosed in 1980-90 were included. Using polymerase chain reaction/single strand conformation polymorphism (PCR/SSCP) analysis 47 mutations were found distributed in 39 of the tumours (49%). Unexpectedly, the majority of the mutations (29/47; 62%) were found in exon 8, and at sequencing 17 of them showed a 14 bp deletion in codons 287-292, causing formation of a stop codon and accordingly a truncated protein lacking the C-terminal. The majority of the patients with the 14 bp deletion were women (13/17), and it seemed as though certain potential risk factors for carcinoma of the head and neck were less common in this group.

Age Factors↗