T HE terms programmed cell death (PCD) 1 and apoptosis are often used interchangeably to describe a mechanism of cellular demise that is believed to play an important role in a wide variety of physiological situations, and that when dysregulated can contribute to the pathogenesis of many diseases ranging from cancer to AIDS. Recently, the bcl-2 gene has emerged as a critical regulator of PCD in a variety of physiological and pathological contexts.

/pdf/bcl-2-and-the-regulation-of-programmed-cell-death-37l5pmzb00.pdf

Bcl-2 and the regulation of programmed cell death

The genome of the Kaposi sarcoma-associated herpesvirus (KSHV or HHV8) was mapped with cosmid and phage genomic libraries from the BC-1 cell line. Its nucleotide sequence was determined except for a 3-kb region at the right end of the genome that was refractory to cloning. The BC-1 KSHV genome consists of a 140.5-kb-long unique coding region flanked by multiple G + C-rich 801-bp terminal repeat sequences. A genomic duplication that apparently arose in the parental tumor is present in this cell culture-derived strain. At least 81 ORFs, including 66 with homology to herpesvirus saimiri ORFs, and 5 internal repeat regions are present in the long unique region. The virus encodes homologs to complement-binding proteins, three cytokines (two macrophage inflammatory proteins and interleukin 6), dihydrofolate reductase, bcl-2, interferon regulatory factors, interleukin 8 receptor, neural cell adhesion molecule-like adhesin, and a D-type cyclin, as well as viral structural and metabolic proteins. Terminal repeat analysis of virus DNA from a KS lesion suggests a monoclonal expansion of KSHV in the KS tumor.

https://www.pnas.org/content/pnas/93/25/14862.full.pdf

Nucleotide sequence of the kaposi sarcoma-associated herpesvirus (hhv8)

Chronic myeloid leukemia (CML) is a clonal myeloproliferative expansion of transformed, primitive hematopoietic progenitor cells. It involves myeloid, monocytic, erythroid, megakaryocytic, B-lymphoid, and occasionally T-lymphoid lineages.1 CML was the first human disease in which a specific abnormality of the karyotype — the Philadelphia (Ph) chromosome — could be linked to pathogenetic events of leukemogenesis.2 It was among the first neoplastic diseases in which therapy with a biologic agent (interferon) was found to suppress the leukemic clone and prolong survival.3 Although heterogeneous, CML is the best-characterized leukemia at a molecular level, and studies in recent years have helped to define further . . .

The biology of chronic myeloid leukemia

Clinical and molecular medicines are undergoing a revolution based on the accelerated advances in biotechnology such as DNA microarrays and proteomics. Answers to fundamental questions such as how does the DNA sequence differ between individuals and what makes one individual more prone for a certain disease are eagerly being sought in this postgenomic era. Several government and nonprofit organizations provide the researchers access to human tissues for molecular studies. The tissues procured by the different organizations may differ with respect to fixation and processing parameters that may affect significantly the molecular profile of the tissues. It is imperative that a prospective investigator be aware of the potential contributing factors before designing a project. The purpose of this review is to provide an overview of the methods of human tissue acquisition, fixation, and preservation. In addition, the parameters of procurement and fixation that affect the quality of the tissues at the molecular level are discussed.

http://molbiol.ru/forums/index.php?act=attach&type=post&id=142562

Effect of fixatives and tissue processing on the content and integrity of nucleic acids.

High expression of the Breast Cancer Resistance Protein (BCRP) gene has been shown to be involved in resistance to chemotherapeutic drugs. Knowledge of the localization of BCRP protein in normal tissues may help unravel the normal function of this protein. Therefore, we characterized the tissue distribution and cellular localization of BCRP in frozen sections of normal human tissues. For this purpose, we used the recently described monoclonal antibody BXP-34 and another independently developed monoclonal antibody directed against BCRP, BXP-21. Both monoclonal antibodies show specific BCRP plasma membrane staining on cytospins obtained from topotecan- or mitoxantrone-selected cell lines, as well as from BCRP-transfected cell lines. Immunoprecipitation experiments using either BXP-21 or BXP-34 yielded a clear M(r) 72,000 BCRP band from BCRP-overexpressing tumor cells. In the topotecan-selected T8 and mitoxantrone-selected MX3 tumor cell lines, BCRP turned out to be differentially glycosylated. In contrast to BXP-34, BXP-21 is able to detect the M(r) 72,000 BCRP protein on immunoblots and is reactive with BCRP in formalin-fixed, paraffin-embedded tissues. Using BXP-21 and BXP-34, prominent staining of BCRP was observed in placental syncytiotrophoblasts, in the epithelium of the small intestine and colon, in the liver canalicular membrane, and in ducts and lobules of the breast. Furthermore, BCRP was present in veinous and capillary endothelium, but not in arterial endothelium in all of the tissues investigated. In the tissues studied, the mRNA levels of BCRP were assessed using reverse transcription-PCR, and these corresponded with the levels of BCRP protein estimated from immunohistochemical staining. The presence of BCRP at the placental syncytiotrophoblasts is consistent with the hypothesis of a protective role of BCRP for the fetus. The apical localization in the epithelium of the small intestine and colon indicates a possible role of BCRP in the regulation of the uptake of p.o. administered BCRP substrates by back-transport of substrate drugs entering from the gut lumen. Therefore, it may be useful to attempt to modulate the uptake of p.o. delivered BCRP substrates, e.g., topotecan or irinotecan, by using a BCRP inhibitor. Clinical trials testing this hypothesis have been initiated in our institute.

https://cancerres.aacrjournals.org/content/canres/61/8/3458.full.pdf

Subcellular localization and distribution of the breast cancer resistance protein transporter in normal human tissues.

Specific amplification of nucleic acid sequences by PCR has been extensively used for the detection of gene rearrangements and gene expression. Although successful amplification of DNA sequences has been carried out with DNA prepared from formalin-fixed, paraffin-embedded (FFPE) tissues, there are only a few reports regarding RNA analysis in this kind of material. We describe a procedure for RNA extraction from different types of FFPE tissues, involving digestion with proteinase K followed by guanidinium-thiocyanate acid phenol extraction and DNase I digestion. These RNA preparations are suitable for PCR analysis of mRNA and even of intronless genes. Furthermore, the universally expressed porphobilinogen deaminase mRNA proved to be useful as a positive control because of the lack of pseudogenes.

An improved strategy and a useful housekeeping gene for RNA analysis from formalin-fixed, paraffin-embedded tissues by PCR.

Expression of bcl-2 in Burkitt's lymphoma cell lines: induction by latent Epstein-Barr virus genes.

Summary. Telomere shortening has been causally linked to replicative senescence in human cells. To characterize telomere-length heterogeneity in peripheral blood cells of normal individuals, we analysed the mean length of telomeric repeat sequences in subpopulations of peripheral blood leucocytes, using fluorescence in situ hybridization and flow cytometry (flow-FISH). Although the telomere length of most haematopoietic subsets was within the same range, the mean telomere length was found to be 15% higher in B compared with T lymphocytes in adult peripheral blood. Whereas telomere loss with ageing corresponded to 33 base pairs (bp) per year in T cells, telomere shortening was slower in B cells, corresponding to 15 bp per year. Separation of adult B-lymphocyte subpopulations based on CD27 expression revealed that telomere length was almost 2 kb longer in CD19+CD27+ (memory) compared with CD19+CD27– (naive) cells. Furthermore, peripheral blood B cells were activated in vitro. Whereas B-cell activation with Staphylococcus aureus Cowan strain (SAC) did not increase telomere length, a striking telomere elongation was observed when cells were stimulated with SAC and interleukin 2 to induce plasma cell differentiation. Our observations support the concept that telomere dynamics in B cells are distinct from other haematopoietic cell lineages and that telomere elongation may play an essential role in the generation of long-term B memory cells.

https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-2141.2002.03910.x

Telomere maintenance in human B lymphocytes

PURPOSETo determine the prevalence of circulating t(14;18)-positive cells in patients with long-term remission after radiation therapy for stage I and II follicular lymphoma.PATIENTS AND METHODSPeripheral-blood mononuclear cells from 21 patients in continuous remission were examined by a two-step polymerase chain reaction (PCR) assay for the detection of cells carrying a t(14;18) translocation with a breakpoint within the major breakpoint region (MBR) or minor cluster region (mcr).RESULTSFollow-up duration was between 25 and 160 months, with a median of 6.5 years. Thirteen patients (62%) showed negative results on repetitive testing. Cells that were t(14;18)-positive were found in eight patients (38%), all carrying a breakpoint in the MBR. One patient relapsed in each group.CONCLUSIONCirculating t(14;18)-positive cells can persist in a high percentage of follicular lymphoma patients in long-term complete remission (CR) after radiation treatment for stage I and II disease. The significance of minimal resid...

Persistence of circulating t(14;18)-positive cells in long-term remission after radiation therapy for localized-stage follicular lymphoma.

Chronic myelogenous leukemia (CML) is associated with a translocation of the BCR and the ABL genes, t(9;22). Results of this event are transcription and translation products that are unique to malignant cells. We therefore designed synthetic ribozymes which are capable of exclusively cleaving the BCR/ABL B3A2-type mRNA without altering normal cellular transcripts. Synthetic B3A2-type transcripts could only be cleaved by B3A2-type mRNA targeted ribozymes and not by any of the controls. The B3A2-type mRNA directed ribozyme, on the other hand, did not cleave any of the control transcripts. The effective delivery of ribonucleic acids by lipofection into K562 cells could be demonstrated by fluorescent microscopy, slot blot analysis, and RNA polyacrylamide gel electrophoresis. In vivo, we were able to induce a significant inhibition of the proliferation of K562 cells with ribozymes directed against the B3A2-type mRNA. Quantitative PCR analyses showed an up to fivefold reduction of the relative number of BCR/ABL mRNA molecules per single cell after exposure to ribozymes compared to controls. We conclude that ribozymes targeted against the B3A2-type BCR/ABL mRNA function in vitro and in vivo.

W. Lange

Papers

An improved strategy and a useful housekeeping gene for RNA analysis from formalin-fixed, paraffin-embedded tissues by PCR.

Expression of bcl-2 in Burkitt's lymphoma cell lines: induction by latent Epstein-Barr virus genes.

Telomere maintenance in human B lymphocytes

Persistence of circulating t(14;18)-positive cells in long-term remission after radiation therapy for localized-stage follicular lymphoma.

In vitro and in vivo effects of synthetic ribozymes targeted against BCR/ABL mRNA.