Frequent loss of the BLID gene in early-onset breast cancer.

The BH3-like motif-containing inducer of cell death (BLID) is an intronless gene localized on 11q24.1. Loss of that region has frequently been reported in early-onset breast cancer and is significantly associated with poor prognosis and reduced survival. Downregulation of BLID is associated with younger age, triple-negative phenotype, and reduced disease-free and overall survival of breast cancer patients. In this study, we investigated allelic loss of BLID in breast tumor specimens from 78 women with invasive breast cancer using 2 dinucleotide polymorphic markers closely linked to the BLID gene (no intragenic marker for BLID is available). Seventy-three cases were informative. Overall, loss of heterozygosity (LOH) at the BLID locus was detected in 32% of the informative cases (23/73). However, in patients 40 years old and younger, LOH was detected in 50% of the cases (9/18). Patients aged 40 years and younger were significantly more likely to experience LOH than those aged 41-55 years (p = 0.04). Specifically, the odds of BLID loss for patients aged 40 years and younger were 3.7 times the odds of loss for patients aged 41-55 years (95% CI, 1.1-13). Our findings suggest a tumor suppressor role of the BLID gene in early-onset breast cancer.

Hodgkin disease: pharmacologic intervention of the CD40-NF kappa B pathway by a protease inhibitor.

The malignant Reed-Sternberg cell of Hodgkin disease is an aberrant B cell that persists in an immunolgically mediated inflammatory infiltrate. Despite its nonproductive immunoglobulin genes, the Reed-Sternberg cell avoids the usual apoptotic fate of defective immune cells through an unknown mechanism. A likely candidate is the surface receptor, CD40, consistently expressed by Reed-Sternberg cells, and the first link in the pathway to NF-kappa B activation, the central regulator of cytokine production and apoptosis. CD40 signaling in B lymphocytes coordinates the immune response, including immunoglobulin isotype switch and Fas-mediated apoptosis. CD40-induced NF-kappa B activation is mediated by adapter proteins, the TNF receptor (TNFR)-associated factors (TRAFs), especially TRAFs 2, 3, and 5. Using a Hodgkin cell line, this study demonstrates that CD40 activation of NF-kappa B is mediated by proteolysis of TRAF3. Results further demonstrate that the pathway can be blocked by treatment with pharmacologic doses of a specific protease inhibitor, pepstatin-A, even in the presence of a mutated NF-kappa B inhibitor, I-kappa B alpha. The stability of TRAF3 regulates CD40/NF-kappa B-mediated control of the immune response, which is central to the biologic activity of the Reed-Sternberg cell. Prevention of TRAF3 proteolysis may be an entry point for design of novel pharmaceuticals to treat Hodgkin disease and immune system disorders. (Blood. 2000;96:2841-2848)

A chromosomal survey of four species of gekkonid lizards.

The karyotypes of the gekkonids were serially graded from the largest to the smallest in the diploid complement. The majority of the chromosomes were subtelocentric. The diploid number was 2n = 38 in Gekko gecko and 2n = 40 in the three species of the genus Hemidactylus examined. A comparative morphometric analysis of the diploid sets of these four members suggested a cytotaxonomic relationship and a possible mode of speciation in this group of reptiles.

Poly(ADP-ribose)-mediated post-translational modification of chromatin-associated human topoisomerase I. Inhibitory effects on catalytic activity.

We have investigated the association of human topoisomerase I with poly(ADP-ribosylated) domains of chromatin and the effects of this modification on the enzyme activity. In vitro poly(ADP-ribosylation) assays demonstrated that this enzyme was one of the major acceptors for this chromatin-dependent post-translational modification. Western blotting procedures using antibody to topoisomerase I indicated that under extensive poly(ADP-ribosylation) conditions, where a majority of poly(ADP-ribose) acceptor molecules form aggregates, the major population of the topoisomerase I associated with chromatin was apparently non-aggregated. The catalytic activity of the topoisomerase I associated with the poly(ADP-ribosylated) chromatin was 3-5-fold inhibited. Additionally, antibody to poly(ADP-ribose) was used to immunofractionate selectively the modified domains of chromatin. Our data suggests the presence of topoisomerase I, both adjacent and distal to the poly(ADP-ribosylated) sites of chromatin. Unmodified and a significant portion of the modified species of enzyme migrated as approximately 100-kDa proteins. However, the modified form of topoisomerase was noted to be catalytically less active as compared to the enzyme bound to the non-poly(ADP-ribosylated) nucleosomes. These results provide evidence, at the cellular level, for the poly(ADP-ribosylation)-mediated regulation of human topoisomerase I and suggest a functional significance for poly(ADP-ribosylation) in topoisomerase-related processes (replication, transcription, and recombination) in eukaryotes.

Sensitivities of NIH/3T3-derived clonal cell lines to ionizing radiation: significance for gene transfer studies.

Rodent cells are frequently used as recipients in experiments involving gene transfer, isolation, and characterization. The present studies were designed to investigate the clonal responses to ionizing radiation of NIH/3T3 cells subjected to DNA-mediated gene transfer. Radiation sensitivity (D0) values were determined for the parental NIH/3T3 cell strain, six clonal cell lines transfected with DNA from radiation-resistant human tumor cells, and six nontransfected clonal cell lines. The radiation sensitivities of four transfected and two nontransfected clonal cell lines differed significantly from parental NIH/3T3 cells (P less than 0.05). Detailed karyotype analysis of two nontransfected clonal cell lines with differing radiation sensitivities showed variation in chromosomal composition. Specifically, a minute chromosome was observed to segregate consistently (in 49 of 50 metaphases) with the genome of one NIH/3T3 clone (D0 2.07 Gy) and was completely absent (from 50 metaphases) in another NIH/3T3 clone (D0 1.06 Gy). In the parental NIH/3T3 strain (D0 2.02 Gy) 10% of cells (3 of 30 metaphases) had such minute chromosomes. These findings demonstrate that the clonal cellular heterogeneity of NIH/3T3 cells is characterized by genotypic and phenotypic variations which must be considered in the experimental design involving gene transfer and expression.

Human epidermal keratinocytes retain radiation resistance following in vitro immortalization and malignant transformation.

The radiobiology of human tumors suggests that multiple factors are involved in clinical radioresponsiveness. To date, no direct experimental evidence is available to correlate intrinsic cellular radiosensitivity with the steps of malignant transformation. We developed an in vitro multistage model of epithelial neoplasia using human epidermal keratinocytes to examine the effects of malignant transformation on radiation response. These cells were first immortalized as a result of infection with a hybrid virus (adenovirus 12 and simian virus 40) and subsequently transformed either by infection with a second virus (Kirsten murine sarcoma virus) or by treatment with a chemical carcinogen (N-methyl-N'-nitro-N-nitrosoguanidine or 4-nitroquinoline-1-oxide). We demonstrate that primary human epidermal keratinocytes were radiation resistant (D0 = 2.24 Gy) as compared with human fibroblasts (D0 = 1.45 Gy) and that this resistance was retained in the immortalized as well as the transformed cell lines. These findings present direct experimental evidence that radiation sensitivity of malignant human keratinocytes is an intrinsic property of the precursor cell that may be conserved through the stages of neoplastic transformation.

Relationship between DNA strand breaks and inhibition of poly (ADP-ribosylation): enhancement of carcinogen-induced transformation.

Inhibition of poly(ADP-Rib) by benzamide (BA) or 3-amino-benzamide (3AB) for a limited period (i.e., when ADP-ribosylation is elevated) during and shortly following X-ray or MNNG-induced DNA damage of BALB/3T3 cells significantly (3- to 30-fold) enhanced transformation frequency by these agents. Individual Type III foci isolated from benzamide, X-ray, or X-ray plus benzamide treated cultures were established and characterized for growth in soft agar and for tumor induction in nude mice. DNA isolated from representative transformed lines established as a result of BA, X-ray or X-ray and BA treatments was transfected onto NIH/3T3 cells. Transformation efficiencies ranging from 0.17 to 0.28 foci/micrograms of DNA were observed suggesting the possibility that dominant transforming gene(s) were responsible for the oncogenic phenotype of radiation and benzamide transformed DNA.

The association of human c-Ha-ras sequences with chromatin and nuclear proteins.

As a step towards the understanding of possible relationship between chromatin organization and regulation of the oncogene expression, we have investigated the chromatin structure of one of the more frequently activated oncogenes, c-Ha-ras, in HeLa-S3 cells. This was accomplished by isolation of the chromatin fractions (soluble and insoluble) after micrococcal nuclease digestion of purified nuclei and probing for the distribution of ras sequences. The polynucleosomal fraction was further resolved by sucrose gradient sedimentation. Southern-blot hybridization of the DNA isolated from various fractions yielded following results: (1) c-Ha-ras sequences segregated predominantly in the lysate fraction. (2) Unlike the B-globin (transcriptionally inactive) sequences, ras-H associated chromatin lacked typical nucleosomal packaging. Furthermore, since post-translational modifications of nuclear proteins have been suggested to modulate the nucleosome structure during DNA transcription and replication, ras sequences, in polynucleosomes immunofractionated on anti-poly (ADP-Ribose) Sepharose were also examined. The data suggested that the major class of this oncogene sequence exists in chromatin more distal to the sites of this particular chromatin modification.