The clinical syndrome of generalized, compensated glucocorticoid resistance is characterized by increased cortisol secretion without clinical evidence of hyper- or hypocortisolism, and manifestations of androgen and/or mineralocorticoid excess. This condition results from partial failure of the glucocorticoid receptor (GR) to modulate transcription of its target genes. We studied the molecular mechanisms of this syndrome in a Dutch kindred, whose affected members had hypercortisolism and approximately half of normal GRs, and whose proband was a young woman with manifestations of hyperandrogenism. Using the polymerase chain reaction to amplify and sequence each of the nine exons of the GR gene alpha, along with their 5'- and 3'-flanking regions, we identified a 4-base deletion at the 3'-boundary of exon 6 in one GR allele (delta 4), which removed a donor splice site in all three affected members studied. In contrast, the sequence of exon 6 in the two unaffected siblings was normal. A single nucleotide substitution causing an amino acid substitution in the amino terminal domain of the GR (asparagine to serine, codon 363) was also discovered in exon 2 of the other allele (G1220) in the proband, in one of her affected brothers and in her unaffected sister. The functional importance of this mutation was tested in a cotransfection study using the recombinant expression vector pRShGR-Ser363 and the glucocorticoid responsive vector mouse mammary tumor virus-chloramphenicol transferase. This amino acid substitution did not alter the function of the glucocorticoid receptor. Using reverse transcription-polymerase chain reaction we could only identify messenger RNA transcripts of the G1220-allele but not of the delta 4-allele in the affected members of this family who were heterozygous for the G1220 mutation. This deletion in the glucocorticoid receptor gene was, thus, associated with the expression of only one allele and a decrease of GR protein by 50% in affected members of this glucocorticoid resistant family. The mutation identified in exon 2 did not segregate with the disease and appears to be of no functional significance. The presence of the null allele was apparently compensated for by increased cortisol production at the expense of concurrent hyperandrogenism.

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BackgroundEpigenetic alterations and loss of heterozygosity are mechanisms of tumor suppressor gene inactivation. A new carcinogenic pathway, targeting the RAS effectors has recently been documented. RASSF1A, on 3p21.3, and NORE1A, on 1q32.1, are among the most important, representative RAS effectors.MethodsWe screened the 3p21 locus for the loss of heterozygosity and the hypermethylation status of RASSF1A, NORE1A and BLU (the latter located at 3p21.3) in 41 neuroblastic tumors. The statistical relationship of these data was correlated with CASP8 hypermethylation. The expression levels of these genes, in cell lines, were analyzed by RT-PCR.ResultsLoss of heterozygosity and microsatellite instability at 3p21 were detected in 14% of the analyzed tumors. Methylation was different for tumors and cell lines (tumors: 83% in RASSF1A, 3% in NORE1A, 8% in BLU and 60% in CASP8; cell lines: 100% in RASSF1A, 50% in NORE1A, 66% in BLU and 92% in CASP8). In cell lines, a correlation with lack of expression was evident for RASSF1A, but less clear for NORE1A, BLU and CASP8. We could only demonstrate a statistically significant association between hypermethylation of RASSF1A and hypermethylation of CASP8, while no association with MYCN amplification, 1p deletion, and/or aggressive histological pattern of the tumor was demonstrated.Conclusion1) LOH at 3p21 appears in a small percentage of neuroblastomas, indicating that a candidate tumor suppressor gene of neuroblastic tumors is not located in this region.2) Promoter hypermethylation of RASSF1A and CASP8 occurs at a high frequency in neuroblastomas.

A novel series of fourteen substituted selenadiazoles has been synthesized and the compounds tested for their in vitro antiproliferative and cytotoxic activities. The tests were carried out against leukemia (CCRF-CEM), colon (HT-29), lung (HTB-54), and breast (MCF-7) cancer cells. In order to assess the selectivity of the compounds under investigation the assays were also carried out on two non-tumoral lines - one mammary (184B5) and one bronchial epithelium (BEAS-2B) cell line. Assay-based antiproliferative activity studies revealed that seven derivatives (2a, 2c, 2e, 2f, 2g, 3a, and 3b) exhibited good activity against MCF-7 cells: for instance, 2c and 2f inhibited cell growth with nanomolar GI₅₀ values. Compound 2f had a better antitumoral profile than vinorelbine and paclitaxel, two drugs that are used as first-line treatments in advanced, recurrent, and/or metastatic cancer. In the other cell lines the compounds showed moderate activity or were inactive - with the exception of 2a, which was also found to have antiproliferative activity. Modulation of the cell cycle and apoptotic effects of active compounds were further evaluated in MCF-7 cells. Of these, 6-bromo[1,2,5]selenadiazolo[3,4-b]pyridine (2a) was the most active, with an apoptogenic effect 3.9 times higher than that of camptothecin, which was used as a positive control. Compound 2a also provoked cell cycle arrest with a significant decrease in the G₀/G₁ phase cell population and an increase in S and G₂/M cells, thus suggesting mitotic arrest prior to metaphase.

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The synthesis and cytotoxic activity of a series of twenty six aroyl and heteroaroyl selenylacetic acid derivatives of general formula Ar-CO-Se-CH2-COOH or Heterar-CO-Se-CH2-COOH are reported. The synthesis was carried out by reaction of acyl chlorides with sodium hydrogen selenide, prepared in situ, and this led to the formation of sodium aroylselenides that subsequently reacted with α-bromoacetic acid to produce the corresponding selenylacetic acid derivatives. All of the compounds were tested against a prostate cancer cell line (PC-3) and some of the more active compounds were assessed against a panel of four human cancer cell lines (CCRF-CEM, HTB-54, HT-29, MCF-7) and one mammary gland-derived non-malignant cell line (184B5). Some of the compounds exhibited remarkable cytotoxic and antiproliferative activities against MCF-7 and PC-3 that were higher than those of the reference compounds doxorubicin and etoposide, respectively. For example, in MCF-7 when Ar = phenyl, 3,5-dimethoxyphenyl or benzyl the TGI values were 3.69, 4.18 and 6.19 μM. On the other hand, in PC-3 these compounds showed values of 6.8, 4.0 and 2.9 μM. Furthermore, benzoylselenylacetic acid did not provoke apoptosis nor did it perturb the cell cycle in MCF-7.

Recently a linkage study on five Icelandic and two English pedigrees has provided evidence for a dominant gene for schizophrenia on 5q11-13 (ref. 1). In that study, families with bipolar illness were not included. Using the same probes, two similar but independent investigations on one Swedish pedigree and on fifteen Scottish families excluded linkage to schizophrenia. To evaluate whether the susceptibility gene on 5q11-13 is a common cause of schizophrenia in other populations, we examined five affected North American pedigrees using probes to the D5S39, D5S76 and dihydrofolate reductase loci. Two families in the present series had cases of bipolar disorder. We found that linkage can be excluded by multipoint analysis. These results, taken together, suggest that the disease gene on 5q11-13 does not account for most cases of familial schizophrenia.

Background. There is a patent need to better characterize early-stage colorectal cancer (CRC) patients. PD-1 ligand (PD-L1) expression has been proposed as a prognostic factor but yields mixed results in different settings. The Consensus Molecular Subtype (CMS) classification has yet to be integrated into clinical practice. We sought to evaluate the prognostic value of PD-L1 expression overall and within CMS in early-stage colon cancer patients, in the hope of assisting treatment choice in this setting. Methods. Tissue-microarrays were constructed from tumor samples of 162 stage II/III CRC patients. They underwent automatic immunohistochemical staining for PD-L1 and the proposed CMS panel. Primary endpoints were overall survival (OS) and disease-free survival (DFS). Results. PD-L1 expression was significantly and independently associated with better prognosis (HR = 0.46 (0.26–0.82), p = 0.009) and was mostly seen in immune cells of the tumor-related stroma. CMS4 five-folds the risk of mortalitycompared with CMS1 (HR = 5.58 (1.36, 22.0), p = 0.034). In the subgroup CMS2/CMS3 analysis, PD-L1 expression significantly differentiated individuals with better OS (p = 0.004) and DFS (p < 0.001). Conclusions. Our study suggests that PD-L1 expression is an independent prognostic factor in patients with stage II/III colon cancer. Additionally, it successfully differentiates patients with better prognosis in the CMS2/CMS3 group and may prove significant for the clinical relevance of the CMS classification.