Quantitative analysis of promoter hypermethylation in laser-microdissected archival specimens.
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Biopsy↗
Biomedical subjects
R Lilischkis
Publications and source records attributed to R Lilischkis.
[Promoter hypermethylation during tumor progression: quantitative analysis employing kinetic PCR].
The methylation of cytosine residues in the promoter region of tumour-suppressor genes is now widely recognised as an alternative mechanism of gene inactivation in cancer. To address the question whether promoter methylation is an early event in the process of malignant transformation and whether quantitative changes are occurring during clonal evolution we developed a new assay for the measurement of cytosine methylation, which could also be applied to laser-microdissected archival specimens. We first improved the bisulfite conversion of genomic DNA in order to analyse cells isolated from archival tissue sections by laser-assisted microdissection. Analysing the methylation of key regulatory genes in archival specimens (n = 38) we could demonstrate that aberrant promoter hypermethylation is an early event during breast cancer development and changes quantitatively and in a gene-specific manner during progression. This novel methodological approach allows the morphology-guided quantitative analysis of changes in the methylation pattern during the clonal evolution of tumour cells. Quantitative differences detected by this assay will offer new insights into early steps in carcinogenesis and tumour cell heterogeneity.
DNA Methylation↗
[Identification of new substrates of cyclin-activated kinases in breast cancers].
Mitogenic and growth-inhibitory signals influence cell-cycle progression through their action on a family of cyclin-dependent kinases (cdks). The activity of cdk complexes is regulated in part by the association of a cyclin partner that acts as a positive effector. These cyclin/CDK complexes promote cell cycle progression by the phosphorylation of key substrates. Cyclin D1 and E are frequently overexpressed in breast cancers and cyclin E overexpression has been correlated with a poor prognostic outcome. The in vivo substrates of cyclin E/CDK2 are, however, not well defined. We screened for cyclin E/CDK2 substrates in nuclear extracts of breast cancer cells as well as using a spotted-array protein library with purified active cyclin E/CDK2 complexes that were expressed in and purified from insect cells. Using this technique several potential cyclin E/CDK2 substrates were isolated. Further work is presently underway to identify these substrates and verify their authenticity as in vivo substrates of cyclin E/CDK2. These potential new substrates will help to unravel the highly complex mechanisms of cell cycle control and perhaps offer new targets for the diagnosis and/or treatment of breast cancer patients.
Breast Neoplasms↗
Positive display of methylated sites: a novel method for the detection of promoter methylation.
Promoter methylation represents an important mechanism for silencing gene expression in higher eukaryotes. To study methylation of the promoter of the tumor suppressor p16INK4a, a fast and simple method was developed that, in contrast to previous studies, relies on the positive display of methylated sites (PDM). The method is based on bisulfite treatment of DNA, polymerase chain reaction (PCR)-amplification of the modified DNA and restriction digest of de novo created restriction sites to positively display DNA methylation in a background of unmethylated DNA. Since methylated as well as unmethylated DNA is amplified, information on the proportion of both is provided. Using this approach, 33 ductal invasive carcinomas, 4 normal mammary tissues, and 4 cell lines were analyzed for methylation. Methylation in the p16INK4a promoter was detected in 1 of 33 carcinomas (3%) and in 0 of 4 normal tissues. The conclusion is that PDM provides a useful tool in determining the degree and pattern of promoter methylation and is suitable to screen large series of tissue samples.
Base Pairing↗
Measles virus-induced immunosuppression in vitro is associated with deregulation of G1 cell cycle control proteins.
Virus-induced immunosuppression is the major cause of the high morbidity/mortality rates associated with acute measles. It has been shown previously that mitogen-dependent proliferation of peripheral blood lymphocytes (PBL) was strongly impaired after contact with the measles virus (MV) glycoproteins F and H expressed on the surface of infected cells, cells transfected with the corresponding expression constructs or UV-inactivated MV (UV-MV). The state of unresponsiveness was not associated with the induction of apoptosis, and a significant proportion of PBL was found to be arrested in the G0/G1 phase of the cell cycle. It is now shown that cell cycle cessation, rather than complete arrest, is induced after MV glycoprotein contact. No obvious role was found for p53 in the induction of this unresponsiveness. With UV-MV as effector, downregulation of p27, an inhibitor of cyclin-dependent kinase (CDK)-cyclin complexes, was significantly delayed after mitogenic stimulation of human PBL. The activities of both CDK4/6-cyclin D and CDK2-cyclin E complexes for phosphorylation of exogenous substrates in vitro were strongly reduced. CDK4, CDK6, cyclins D3 and E and, to a minor extent, CDK2 failed to accumulate at the protein level after mitogenic stimulation in the presence of UV-MV. These data indicate that MV-induced proliferative unresponsiveness of PBL to mitogenic stimulation is associated with a drastic deregulation of the expression of cell cycle genes essential for the G1/S phase transition.
Adult↗
Differential phosphorylation of T-47D human breast cancer cell substrates by D1-, D3-, E-, and A-type cyclin-CDK complexes.
The cyclin-dependent kinases (CDKs) promote cell cycle transitions in mammalian cells by phosphorylation of key substrates. To characterize substrates of the G1 and S phase cyclin-CDK complexes, including cyclin D1-CDK4, cyclin D3-CDK4, cyclin D3-CDK6, cyclin E-CDK2, and cyclin A-CDK2, which are largely undefined, we phosphorylated T-47D breast cancer cell nuclear lysates partially purified by ion-exchange chromatography with purified baculovirus expressed cyclin-CDK complexes. A comparison of the substrates that were phosphorylated by the different cyclin D-CDKs revealed some common as well as specific substrates. Hence, cyclin D1-CDK4 specifically phosphorylated a 38-kDa protein while cyclin D3-CDK4 specifically phosphorylated proteins of 105, 102, and 42 kDa. A 24-kDa protein was phosphorylated by both complexes. Cyclin D3-CDK6 exhibited similar substrate preferences to cyclin D3-CDK4, phosphorylating the 105- and 102-kDa proteins but not the 24-kDa protein. Hence, both the cyclin D1 and D3 as well as CDK4 and CDK6 subunits can confer substrate specificity on the overall cyclin D-CDK complex. Cyclin E-CDK2 and cyclin A-CDK2 phosphorylated a greater number of substrates than the cyclin D-CDKs, ranging in size from 10 kDa to over 200 kDa. Twenty-two substrates were common to both complexes, while six were specific for cyclin A-CDK2 and only one protein of 34 kDa was specific for cyclin E-CDK2. These studies indicate that cyclins E and A modulate the specificity of CDK2 and have demonstrated substrates that may be important for the specific roles of these cyclin-CDKs during G1 and S phase progression. Protein sequencing of one of the cyclin-CDK substrates characterized in this study identified this protein as nucleolin, a previously characterized CDC2 (CDK1) substrate, thus indicating the utility of this approach in identifying cyclin-CDK targets. These results show that both the cyclin and CDK subunits can regulate the substrate specificity of the overall cyclin-CDK complex and have demonstrated numerous substrates of D-, E-, and A-type cyclin-CDK complexes potentially involved in regulating transit through the G1 and S phases of the cell cycle.
Amino Acid Sequence↗
[Clonal analysis in cells using PCR and laser microdissection].
Clonality represents one of the hallmarks of neoplastic cell growth. X-chromosomal inactivation patterns have been used to determine clonality in various tumors. This approach is limited by admixture of polyclonal non-tumor stroma cells among which the monoclonal proliferation may be missed. In order to overcome this limitation, we combined a sensitive PCR based DNA analysis with a highly selective microdissection technique using a laser beam. In sections of intraductal mammary carcinomas tumor cell complexes of at least 100 cells were isolated by removing the surrounding stroma by laser irradiation. Thereby, tumor cells could be isolated without contaminating non-neoplastic elements. Clonality in these cells was determined using two X-chromosomal polymorphic sites-phosphoglycerate kinase 1 (PGK1) and human androgen receptor (HUMARA). Control experiments could show the polyclonal nature of the surrounding tissue. Moreover, complete destruction of DNA by laser irradiation was assured. The technique requires a certain amount of cells and DNA in order to avoid artefacts that result from preferential amplification of exclusively one X-chromosomal allele in small samples. We conclude that combination of laser-microdissection with PCR analysis of X-chromosomal inactivation patterns enables the detection of clonal cell populations in heterogeneous tissues. Studies of clonality in borderline cases between reactive and neoplastic proliferations or premalignant lesions are made possible by this technique.
Breast Neoplasms↗
Cancer-associated mis-sense and deletion mutations impair p16INK4 CDK inhibitory activity.
The p16INK4 gene is a candidate tumour-suppressor gene which maps to the genomic locus 9p21, and mutations of this gene are associated with melanoma and other cancers. Biochemical studies suggest that p16INK4 mediates its effects by specifically inhibiting the G1 cyclin-dependent kinases CDK4 and CDK6, thereby regulating the progression through G1 into S phase of the cell cycle. To evaluate the functional effects of mutations in p16INK4 which have been observed in primary cancers and cancer cell lines, we constructed a series of deletion mutants comprising amino acid regions 9-72, 9-131, 73-131 and 73-156; a mis-sense mutation identified in melanoma (Arg87Pro); and the polymorphism Ala48Thr and investigated their ability to inhibit cyclin D1/CDK4 kinase activity in vitro. Removal of 25 amino acids from the carboxy terminus of p16INIK4 (9-131) had little impact on its inhibitory activity. In contrast, deletion of the 65 N-terminal amino acids comprising the first and second ankyrin repeats of p16INK4 (73-131) abolished its inhibitory activity. The carboxy (73-156) and amino termial (9-72) fragments of p16INK4 also failed to inhibit cyclin D1/CDK4 activity. These results indicate that the core region (73-131) as well as amino acids N-terminal of this sequence are important, whereas sequences C-terminal of amino acid 131 are less important for the inhibitory activity of this molecule. The melanoma-associated Arg87Pro mutation resulted in loss of inhibitory activity, whereas the Ala148Thr polymorphic variant was as effective as the alanine variant of p16INK4 in inhibiting D1/CDK4 kinase activity. Binding assays revealed that inhibition was invariably associated with p16INK4 binding to CDK4. Hence, our studies indicate that minor perturbations in p16INK4 primary structure can lead to loss of its inhibitory activity, possibly contributing to oncogenesis in numerous cell types.
Amino Acid Sequence↗
Expression of the cyclin-dependent kinase inhibitors p16INK4, p15INK4B and p21WAF1/CIP1 in human breast cancer.
Aberrant cyclin expression has been implicated in oncogenesis in a number of human cancers. Since altered function of regulators of cyclin-dependent kinase (CDK) activity other than cyclins, in particular CDK inhibitors, might play a similar role in oncogenesis, we examined the expression and regulation of the CDK inhibitors p16INK4, p15INK4B and p21WAF1/CIP1 in human breast cancer cell lines. Both the INK4 and INK4B genes were homozygously deleted in 3 cell lines, while INK4 alone was deleted in 2 cell lines. A further 2 cell lines displayed loss of an allele at this locus, and in 1 of these the remaining allele contained a mis-sense mutation within the coding region of the p16INK4 protein. The majority of cell lines examined, including 2 normal mammary epithelial cell strains, expressed low levels of INK4 mRNA and low or undetectable levels of INK4B mRNA. However, INK4 mRNA was expressed at high levels in 5 cell lines, and this was associated with deletion or inactivation of the retinoblastoma susceptibility gene product pRB but not with mutation of TP53. No deletions of the WAF1/CIP1 gene were observed, but WAF1/CIP1 mRNA levels were reduced in cell lines with TP53 mutation. Transfection of a p16INK4 expression vector into MDA-MB-231 cells lacking the INK4 gene failed to produce any p16INK4-expressing cell lines, suggesting that such cells were selected against in continuous culture. Despite the frequent deletion of INK4 in breast cancer cell lines, no evidence was obtained for INK4 deletions in DNA from 45 primary breast carcinomas. Thus, homozygous deletion of the INK4 gene appears to be a rare event in primary breast cancer.
Base Sequence↗
Isolation of monocytic serine esterase and evaluation of its proteolytic activity.
Monocytes are characterized by high activity of alpha-naphthyl acetate esterase (ANAE), distinguishing them from all other blood cells. The physiological function of this monocyte marker enzyme has not yet been elucidated. In this study ANAE's potential proteolytic activity was analyzed because serine esterases/proteases can function as effector molecules in cell-mediated cytotoxicity and because monocytes-macrophages are known to exert cytotoxic effects on tumor cells. This enzyme was purified from the monocytic cell line U-937 by preparative isoelectric focusing and a three-step high-performance liquid chromatography that conserved its catalytic activity. It has a molecular mass of 60 kd, and partial amino acid sequence revealed that the enzyme is not identical to known serine esterases/proteases. The purified enzyme failed to digest a couple of peptides, indicating lack of protease activity. In addition, the esterolytic activity of ANAE was not inhibited by protease inhibitors. The isolation and purification of ANAE enable further studies concerning its function in monocytes-macrophages and its relation to monocytic cytotoxicity.
Amino Acid Sequence↗
Salmonella in snakes of the Gran Chaco, Paraguay. Description of the "new" flagellar antigen RZ72.
Twenty-three snakes of nine different species were investigated. Of these, 21 were carriers of a total of 22 different Salmonella serovars (subspecies I 14 types, subspecies IIIb 6 types, subspecies IV 2 types). A triphasic variant of S. IIIb 48:i:z:Rz72 was identified for the first time. Numerous serovars of subspecies I strains have previously been demonstrated in humans and animals in large areas of South America. It is concluded that these snakes may constitute a potential reservoir for human infections. Biochemically variant strains occurred as a lactose-positive variant of S. michigan, an indole-positive isolate of S. IIIb 61:r:z53 as well as a dulcitol-positive strain of S. IIIb 48:i:z:Rz72. The method of preparation of the specific diagnostic Rz72 factor serum for determination of this antigen is described.
Animals↗