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.

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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)

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.

Publisher Summary This chapter focuses on the proteins of the Myc network that are essential regulators of cell growth and differentiation. The identification of the Myc partner, Max, in 1991 and the subsequent realization that this protein is the essential dimeric partner for all known c-Myc functions was a major boost to the field and led to a number of very interesting observations and findings. The chapter focuses on c-Myc's role as a transcription factor in the regulation of cell growth, apoptosis, and transformation. The most exciting recent findings suggest that the Myc network not only includes proto-oncoproteins (c-, N-, and L-Myc) but, with the Mad family proteins, also potential tumor suppressors. This together with the fact that Myc proteins as well as Max are essential, as deduced from homozygous disruption of the genes in mice, places the Myc network in a central position in the regulation of cell growth and homeostasis. Genes that have been generated by the duplication of and divergence from an ancestral gene(s) are grouped into families. The myc family of protooncogenes has most likely arisen through such duplications. It currently consists of three well-characterized members; c-myc, N-myc, and L-myc. Two additional genes, B-myc and S-myc, have been identified only in rodents. The c-, N-, and L-myc genes share similar genomic organization and the corresponding proteins contain several regions of high sequence homology. The identification of the Myc dimerization partner Max has significantly advanced our understanding of the molecular function of c-Myc.

Proteins of the Myc Network: Essential Regulators of Cell Growth and Differentiation

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.

R Fritzen

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.