Glutathione S-transferase isoenzymes in human lung tumors

In the present studies we have compared the levels of glutathione(GSH) and GSH-related enzymes in lung tumors and correspondingnormal tissues obtained from the same individuals. We have alsoimmunologically quantitated the relative amounts of glutathioneS-transferase (or GST-P) type antigen in tumors and adjacentnormal tissues from five patients. GST activities towards 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid were found to beelevated in tumors from two out of five patients (patients #1and 4), whereas the activity towards these substrates was markedlysuppressed in the tumor tissue from one of the patients (#5).Immunotitration and Western blot studies using antibodies raisedagainst -type GST isoenzymes of human lung and placenta indicatedinduction of GST -type isoenzyme in tumors from patients #1and 4 and suppression of this isoenzyme in tumor from patient#5. The tumors from patients #2 and 3 did not show any increasein GST activity or GST -type antigen. Except for the tumor frompatient #5, the GSH content was higher in the tumors from otherpatients. GSH reductase activity was found to be elevated intumors of all the patients examined in this study. These resultsindicate that GSH and GSH related enzymes are differentiallyaltered in lung tumors and GSH levels and GST - or GST-P-typeisoenzyme(s) are not uniformly elevated in all tumors.     

Epidermal growth factor receptor regulates normal urothelial regeneration

Members of the epidermal growth factor (EGF) family and their receptors are involved in many cellular processes, including proliferation, migration, and differentiation. We have previously reported that these growth factors are expressed and have specific regulatory functions in an organ-like culture model of normal human urothelial cells. Here, we used this model to investigate the involvement of EGF receptor (EGFR) in human urothelial regeneration. Three 4-mm-diameter damaged areas were made in confluent normal human urothelial cell cultures with a biopsy punch. Regeneration was measured, on fixed stained cultures, with an image analyzer, at 4, 24, and 48 hours after injury. Cell proliferation was assessed by 5-bromo-2-deoxyuridine incorporation. To identify EGF family factors potentially involved in the healing process, we studied the effect of these factors on damaged confluent cultures and the level of expression of mRNAs extracted from these cultures. EGFR inhibition of the proliferation and migration of urothelial cells was tested with (1). a specific tyrosine kinase inhibitor (AG1478) and (2). a blocking anti-EGFR antibody (LA22). Exogenously added amphiregulin, EGF, transforming growth factor-alpha and heparin-binding EGF (HB-EGF) stimulated urothelial regeneration. The damaged areas were repaired by regrowth within 48 hours. Both AG1478 and LA22 inhibited the repair (by 50% and 30%, respectively), as well as proliferation and migration. This regeneration was accompanied by increased HB-EGF mRNA expression in cultures of cells from four of six subjects, but no corresponding change in EGFR protein level was observed. These results indicate that the EGFR signaling pathway is involved in urothelial regeneration. Our data support an autocrine role of HB-EGF in this process and suggest that the EGFR pathway is a potential therapeutic target for modulating urothelial cell proliferation.     

ETV6/RUNX1 fusion at diagnosis and relapse: some prognostic indications

This study was undertaken in order to compare the interphase and metaphase cytogenetics of 28 patients with ETV6/RUNX1 positive acute lymphoblastic leukemia, at diagnosis and relapse. The median time to relapse was 26 months. The significant fusion positive population heterogeneity revealed at interphase by a commercial probe for ETV6/RUNX1 fusion has not been described before. Six diagnostic samples had a single abnormal population; others had up to five each, which differed in the numbers of RUNX1 signals, and in the retention or loss of the second ETV6 signal. In contrast, the number of fusion signals was more constant. At relapse, there were fewer populations; the largest or unique clone was sometimes a re-emergence of a minor, diagnostic one, with a retained copy of ETV6 and the most RUNX1 signals. Abnormal, fusion negative clones were identified in bone marrow samples at extra-medullary relapse. Variant three or four-way translocations, which involved chromosomes 12 and 21, were prominent among the complex rearrangements revealed by metaphase FISH. The frequency of their occurrence at diagnosis and reappearance at relapse, sometimes accompanied by minor clonal evolution, was another new observation. Other recurrent cytogenetic features included a second copy of the fusion signal in six cases, partial duplication of the long arm of the X chromosome in two cases, and trisomy 10 in three cases. In comparing our data with previously reported cases, a picture is beginning to emerge of certain diagnostic features, which may provide circumstantial evidence of an increased risk of relapse.     

Novel mutations in the EXT1 gene in two consanguineous families affected with multiple hereditary exostoses (familial osteochondromatosis)

Multiple hereditary exostoses (HME) is an autosomal dominant developmental disorder exhibiting multiple osteocartilaginous bone tumors that generally arise near the ends of growing long bones. Here, we report two large consanguineous families from Pakistan, who display the typical features of HME. Affected individuals also show a previously unreported feature--bilateral overriding of single toes. Analysis using microsatellite markers for each of the known EXT loci, EXT1, EXT2, and EXT3 showed linkage to EXT1. In the first family, mutation analysis of the EXT1 gene revealed that affected individuals were heterozygous for an in-frame G-to-C transversion at the conserved splice donor site in intron 1. This mutation is predicted to disrupt splicing of the first intron and produce a frameshift that leads to a premature termination codon. In the second family, an insertion of an A in exon 8 is predicted to produce a frameshift at codon 555 followed by a premature termination, a further 10 codons downstream. In both families, an increased number of affected male subjects were observed. In affected females in family 2, phenotypic variability and incomplete penetrance were noted.     

A novel autosomal recessive non-syndromic deafness locus (DFNB35) maps to 14q24.1-14q24.3 in large consanguineous kindred from Pakistan

Autosomal recessive nonsyndromic deafness is one of the most frequent forms of inherited hearing impairment. Over 30 autosomal recessive nonsyndromic hearing loss loci have been mapped, and 15 genes have been isolated. Of the over 30 reported autosomal recessive nonsyndromic hearing loss (NSHL) loci, the typical phenotype is prelingual non-progressive severe to profound hearing loss with the exception of DFNB8, which displays postlingual onset and DFNB13, which is progressive. In this report we describe a large inbred kindred from a remote area of Pakistan, comprising six generations and segregating autosomal recessive nonsyndromic prelingual deafness. DNA samples from 24 individuals were used for genome wide screen and fine mapping. Linkage analysis indicates that in this family the NSHL locus, (DFNB35) maps to a 17.54 cM region on chromosome 14 flanked by markers D14S57 and D14S59. Examination of haplotypes reveals a region that is homozygous for 11.75 cM spanning between markers D14S588 and D14S59. A maximum two-point LOD score of 5.3 and multipoint LOD score of 7.6 was obtained at marker D14S53. The interval for DFNB35 does not overlap with the regions for DFNA9, DFNA23 or DFNB5.