An association between inflammation and cancer has long been recognized, but the cause and effect relationship linking the two remains unclear. Myc is a pleiotropic transcription factor that is overexpressed in many human cancers and instructs many extracellular aspects of the tumor tissue phenotype, including remodeling of tumor stroma and angiogenesis. Here we show in a beta-cell tumor model that activation of Myc in vivo triggers rapid recruitment of mast cells to the tumor site-a recruitment that is absolutely required for macroscopic tumor expansion. In addition, treatment of established beta-cell tumors with a mast cell inhibitor rapidly triggers hypoxia and cell death of tumor and endothelial cells. Inhibitors of mast cell function may therefore prove therapeutically useful in restraining expansion and survival of pancreatic and other cancers.

Based on the high incidence of loss of heterozygosity for loci on chromosome 17p in the vicinity of the p53 locus in human breast tumors, we investigated the frequency and effects of mutations in the p53 tumor suppressor gene in mammary neoplasia. We examined the p53 gene in 20 breast cancer cell lines and 59 primary breast tumors. Northern blot analysis, immunoprecipitation, and nucleotide sequencing analysis revealed aberrant mRNA expression, overexpression of protein, and point mutations in the p53 gene in 50% of the cell lines tested. A multiplex PCR assay was developed to search for deletions in the p53 genomic locus.

Genetic alpha-tryptase deficiency is common and varies strikingly between ethnic groups. Because beta-tryptases are implicated in allergic disorders, inherited differences in alpha/beta-genotype may affect disease susceptibility, severity and response to tryptase inhibitor therapy.

Research in the last 3 years has made it increasingly clear that a large variety of human tumors contain mutated p53 tumor suppressor genes (reviewed in ref. 1). Since mutations are most often distributed over at least 5 exons (exons 5-9) spanning 2.9 kb of coding sequence, faster methods for scanning the region have been developed. ~2)A popular method for detection of point mutations and deletions in the p53 gene is single-stranded conformation polymorphism (SSCP). (2'3) The technique is based on altered migration speeds through solid supports of singlestranded DNA fragments carrying mutations. The altered migration of DNA is presumably caused by differences in the conformation of single-stranded DNA. The method depends heavily on experimental conditions that optimize migration differences of the conformation polymorphs. Thus, adding glycerol to the polyacrylamide, reducing the temperature, increasing the length of the run, and so forth result in a greater level of reproducibility. ~2' 3~ In spite of these modifications, reproducibility and resolution present recurrent problems using polyacrylamide as a gel support, presenting the need for novel polymers for this purpose. There is also a need to develop a simple nonradioactive technique for screening for mutations in the p53 gene in the clinical laboratory. A gel electrophoresis technique that takes advantage of differences in mobility of wild-type and mutation-bearing DNA fragments could prove useful for this purpose. Double-stranded DNA as heteroduplexes (HTX) migrate at different rates compared to DNA as homoduplexes in solid supports. (4's~ These differences can be visualized easily by staining with ethidium bromide. However, the resolution ability of polyacrylamide gel supports for detection of the two species varies with the sequence of the DNA fragments. ~4' s~In an effort to improve the sensitivity levels of the radioactive SSCP analysis, and to resolve the nonradioactive duplexes, we began to experiment with other polymers. We tested polyacrylamide, Hydrolink-D5000, and Hydrolink-MDE under identical experimental conditions with and without 10% glycerol. We found that Hydrolink-MDE (AT Biochem, Malvern, PA), a vinyl polymer, gave improved resolution of bands as well as reproducibility in the detection of both single-stranded DNA in SSCP and of DNA duplexes in the HTX analyses. Because the SSCP analysis is unable to detect mutations that do not result in polymorphs with altered migrations, a combination of SSCP analysis with HTX analysis might provide more reliable results than either one alone.Human mammary tumors were analyzed by SSCP for mutations in p53 exons 5-9 using 5% polyacrylamide. ~6) A cell line derived from normal breast tissue (HBL-100) was used as a negative control, whereas breast tumor cell lines served as positive controls (SKBR3 and HS578 for exon 5, T47D for exon 6, MW for exon 7, and BT474 for exon 8). The colon cancer cell line, SW480 was used as a positive control for mutation in exon 9 of the p53 gene. It should be noted that thes...

Cultured rat granulosa cells have provided a useful model to examine the hormonal regulation of inhibin secretion. In the present study we have used the cloned rat inhibin alpha- and beta A-subunit cDNAs to characterize the influences of gonadotropins, growth factors, and GnRH on inhibin subunit mRNA levels in granulosa cells obtained from immature estrogen-treated rats. Cells were cultured in medium with or without added hormones. Total RNA from cultured cells was extracted and hybridized with 32P-labeled inhibin alpha- and beta A-subunit cRNA or beta-actin cDNA probes, and inhibin subunit mRNA levels were normalized with beta-actin mRNA levels. Treatment of granulosa cells with FSH increased inhibin alpha- and beta A-subunit mRNA levels in a dose-dependent manner. Similarly, LH, but not PRL, increased alpha- and beta A-subunit mRNA levels in granulosa cells pretreated with FSH to induce functional LH and PRL receptors. The effects of FSH and LH on inhibin subunit mRNA levels were mimicked by forskolin, which increased alpha- and beta A-subunit transcripts in a dose- and time-dependent manner, suggesting involvement of the cAMP-dependent protein kinase-A pathway. Since several growth factors have been shown to influence inhibin secretion, their effects on inhibin subunit mRNA levels were also studied. Treatment of cells with transforming growth factor-beta 1 increased both basal and FSH-stimulated inhibin alpha- and beta A-subunit mRNA content, whereas insulin-like growth factor-I had no significant effect. In contrast, both epidermal growth factor (EGF) and basic fibroblast growth factor (FGF) markedly suppressed both basal and FSH-stimulated inhibin subunit transcript levels. The inhibitory effects of EGF and basic FGF were dose dependent and persisted from 12-72 h of incubation. The regulatory peptide GnRH, which decreases inhibin secretion, was also found to suppress FSH-stimulated inhibin alpha- and beta A-subunit mRNA levels in a dose-dependent manner. Furthermore, the effects of GnRH could be counteracted by coincubation with a GnRH antagonist, suggesting the involvement of specific GnRH-binding sites in GnRH action. These studies indicate that, except for insulin-like growth factor-I, the effects of gonadotropins, growth factors (EGF, basic FGF, and transforming growth factor-beta 1), and GnRH on inhibin secretion are related to their regulation of inhibin alpha- and beta A-subunit mRNA levels.

Recent reports suggest that activin (the dimer of inhibin beta subunits with FSH-releasing activity) has specific receptors on ovarian granulosa cells. The present study examined the effects of purified porcine activin on inhibin secretion and mRNA levels in granulosa cells obtained from immature, estrogen-treated rats. Cells were cultured for 48 h in culture media, or media containing FSH (10 ng/ml) and/or activin (30 ng/ml). Western blot analyses performed with affinity-purified antisera to inhibin alpha- and beta A-subunits revealed that treatment with either FSH or activin increased the secretion of inhibin alpha beta dimer (Mr 30,000), with a further increase after cotreatment. These results were confirmed by an inhibin alpha-subunit RIA, which revealed 7-, 14-, and 71-fold increases in the secretion of immunoreactive inhibin-alpha by activin, FSH, and activin plus FSH, respectively. TGF beta, a structural homolog of activin, also stimulated inhibin release, whereas follistatin was ineffective. Total RNA from cultured cells was hybridized with 32P-labeled inhibin alpha-subunit cRNA or beta-actin cDNA probes, and inhibin-alpha message levels were normalized with beta-actin mRNA levels. Northern blot analysis revealed that treatment with FSH and activin increased hybridization of a 1.5 kilobase (kb) message, corresponding to the inhibin alpha-subunit mRNA. Slot blot analyses indicated a 6- and 8-fold stimulation of inhibin alpha-subunit mRNA levels by FSH and activin, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum mast cell tryptase levels are used as a diagnostic criterion and surrogate marker of disease severity in mastocytosis. Approximately 29% of the healthy population lacks α tryptase genes; however, it is not known whether lack of α tryptase genes leads to variability in tryptase levels or impacts on disease severity in mastocytosis. We have thus analyzed tryptase haplotype in patients with mastocytosis, computing correlations between haplotype and plasma total and mature tryptase levels; and disease category. We found: (1) the distribution of tryptase haplotype in patients with mastocytosis appeared consistent with Harvey-Weinberg equilibrium and the distribution in the general population;(2) the disease severity and plasma tryptase levels were not affected by the number of α or β tryptase alleles in this study; and (3) information about the tryptase haplotype did not provide any prognostic value about the severity of disease. Total and mature tryptase levels positively correlated with disease severity, as well as prothrombin time and partial thromboplastin time, and negatively correlated with the hemoglobin concentration.

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