Common expression of a tumor necrosis factor resistance mechanism among gynecological malignancies

Summary The efficacy of tumor necrosis factor (TNF) as an anticancer agent is limited. This limitation might be related to the expression of a protein-synthesis-dependent resistance mechanism that prevents the lysis of tumor cells by TNF. To test this possibility eight randomly selected human cell lines, three derived from ovarian carcinomas and five derived from cervical carcinomas, were tested for their in vitro sensitivity to TNF-mediated lysis. The results of this analysis showed that all eight cell lines are normally resistant to lysis by TNF. However, in the presence of inhibitors of protein synthesis, seven of them showed a significant increase in TNF-mediated lysis. Measurement of protein synthesis showed that there is a linear correlation between the level of inhibition of protein synthesis and the level of TNF-mediated lysis. The fact that seven of eight randomly selected cell lines are resistant to TNF because they express a protein-synthesis-dependent resistance mechanism suggests that this mechanism of resistance may be common among gynecological cancers. The results also suggest that a therapy involving TNF and inhibitors of protein synthesis might be useful for the treatment of gynecological malignancies.     

DNA sequencing separations in capillary gels on a modified commercial DNA sequencing instrument

DNA sequencing separations of standard DNA fragments of known sequence have been achieved in small diameter capillary gels electrophoresed and analyzed in parallel in a modified commercial DNA sequencer instrument. DNA sequencing in terms of base-calling accuracy is comparable to conventional slab gels; however, the separations in the capillary were performed somewhat faster and required less sample than those in the slab gel. Advantages of this approach vs. separations on conventional slab gels are discussed.     

Localization of 27 DNA markers to the region of human chromosome 22q11-pter deleted in patients with the DiGeorge syndrome and duplicated in the der22 syndrome

DiGeorge syndrome is a human developmental field defect with the pathological features of an abnormality of embryogenesis at 4 to 6 weeks of gestation. Cytogenetic analyses of patients have revealed a number of instances of monosomy 22q11-pter in this condition. We have analyzed 52 DNA markers that map to 22q11-pter and have found 27 that are deleted in DiGeorge syndrome patients with known monosomy for part of this region and that are duplicated in patients with the der22 syndrome. The set of clones mapping to the DiGeorge region was further assigned to a proximal or a distal location within the deletion.     

Human chromosome 17 NotI linking clones and their use in long-range restriction mapping of the Miller-Dieker chromosome region (MDCR) in 17p13.3

A NotI linking library constructed from flow-sorted human chromosome 17 material was screened to aid in construction of a long-range restriction map of the Miller-Dieker chromosome region (MDCR) in 17p13.3. A total of 66 clones were mapped to one of eight regions of chromosome 17 using a somatic cell hybrid panel, and 44/66 (67%) of these clones cross-hybridized to rodent DNA on Southern blots. Of these, 24 clones were tested and all mapped to mouse chromosome 11, the homolog of human chromosome 17. Four linking clones mapped to 17p13.3 and were used for pulsed-field gel electrophoresis studies along with six other anonymous probes previously mapped to this region. Clone L132 was found to be deleted in all Miller-Dieker patients tested (n = 15) and therefore lies within the critical region for this disorder. It detects two NotI fragments (180 and 320 kb), one of which (320 kb) was shared by YNZ22 and YNH37, two probes previously shown to be co-deleted in all patients with the Miller-Dieker syndrome (MDS). These results indicate that all MDS patients share a minimum deletion region of greater than 370 kb. Two other NotI clones, L53 and L125, mapped telomeric to the MDS critical region and share a 600-kb MluI fragment with each other and with YNZ22/YNH37. This provides a 930-kb MluI map that encompasses the distal boundary of the MDS critical region but does not include the proximal boundary. A total of over 2 Mbp is represented in the MluI fragments by probes in subband p13.3, a cytogenetic region estimated to be 3-4 Mbp.     

Bleeding time in patients with hepatic cirrhosis.

OBJECTIVE--To determine the frequency of an abnormal bleeding time in patients with cirrhosis and to relate this to known factors that affect primary haemostasis and to the severity of liver disease. DESIGN--Prospective clinical and laboratory study in patients admitted for complications or investigations of liver disease. SETTING--Royal Free Hospital hepatobiliary and liver transplantation unit. SUBJECTS--100 Consecutive inpatients aged 17-74 with various forms of cirrhosis, including alcoholic, biliary, autoimmune, viral, and cryptogenic. At least 10 days had elapsed since any episodes of bleeding, resolution of sepsis, or alcohol intake. No patient was taking any drug known to affect primary haemostasis. MAIN OUTCOME MEASURES--Bleeding time as measured with the Simplate double blade template device. A bleeding time longer than 10 minutes was considered abnormal. Other measures were platelet count, prothrombin time, partial thromboplastin time, packed cell volume, and blood urea, serum bilirubin, and serum albumin concentrations, all measured on each subject at the same time by standard laboratory methods. RESULTS--A weak but significant correlation existed between the bleeding time and the platelet count (rs = 0.483; p less than 0.001). There were significantly lower platelet counts, longer prothrombin times, and higher blood urea and serum bilirubin concentrations in the 42 patients with bleeding times of 10 minutes or more compared with the 58 patients with bleeding times less than 10 minutes. Multiple linear regression analysis showed that the bilirubin concentration as well as the platelet count was independently correlated with the bleeding time. The combination of a platelet count greater than 80 x 10(9)/l and a prothrombin time less than 17 seconds (usually taken as safe limits for performing routine liver biopsy) did not predict a normal bleeding time. Ten of 39 patients fulfilling these criteria had a prolonged bleeding time. CONCLUSIONS--Prolonged bleeding time is common in patients with cirrhosis, even in those with prothrombin times and platelet counts within "safe limits" for invasive procedures. The severity of liver disease as assessed by the bilirubin concentration plays an important part in determining the bleeding time in cirrhosis. The bleeding time should be measured when assessing patients for invasive procedures who have a raised bilirubin concentration or poor hepatic function, even if the platelet count and prothrombin time are considered adequate.     

Caseload or workload? Scoring complexity of operative procedures as a means of analysing workload.

OBJECTIVE--To present a more realistic assessment of surgical workload than that provided by a case count. DESIGN--Prospective study of all the operative procedures performed in one year, classified according to the British United Provident Association''s schedule of procedures and scored by the "intermediate equivalent" value (taking the recommended fee value of an intermediate operation as 1.0) compared with the number of operations performed. SETTING--General surgical unit of Taunton and Somerset Hospital, comparing four consultant surgeons and their teams. PATIENTS--Inpatients and day patients admitted under the care of general surgeons during 1989. MAIN OUTCOME MEASURE--Difference between the apparent workload represented by simple case counting (caseload) and the actual workload represented by calculation of the total "intermediate equivalent" value. RESULTS--The workload assessed in terms of intermediate equivalent values was greater than that suggested by case counting for complex operations (12% v 4%), operations at the district hospital (82% v 74%), and operations performed by consultants (53% v 35%) and was lower for minor operations (20% v 42%), operations at the community hospitals (18% v 26%), and operations performed by surgeons in training grades and clinical assistants (47% v 66%). CONCLUSIONS--The use of the intermediate equivalent values as an indicator of complexity allows a more realistic assessment of the operative workload than a simple case count of the number of different operations and is recommended for comparing workload in different hospitals and departments.     

Kinds and spectrum of mutations induced by 1-nitrosopyrene adducts during plasmid replication in human cells.

1-Nitropyrene has been shown in bacterial assays to be the principal mutagenic agent in diesel emission particulates. It has also been shown to be mutagenic in human fibroblasts and carcinogenic in animals. To investigate the kinds of mutations induced by this carcinogen and compare them with those induced by a structurally related carcinogen, (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetra-hydrobenzo [a]pyrene (BPDE) (J.-L. Yang, V. M. Maher, and J. J. McCormick, Proc. Natl. Acad. Sci. USA 84:3787-3791, 1987), we treated a shuttle vector with tritiated 1-nitrosopyrene (1-NOP), a carcinogenic mutagenic intermediate metabolite of 1-nitropyrene which forms the same DNA adduct as the parent compound, and introduced the plasmids into a human embryonic kidney cell line, 293, for DNA replication to take place. The treated plasmid, pZ189, carrying a bacterial suppressor tRNA target gene, supF, was allowed 48 h to replicate in the human cells. Progeny plasmids were then rescued, purified, and introduced into bacteria carrying an amber mutation in the beta-galactosidase gene in order to detect those carrying mutations in the supF gene. The frequency of mutants increased in direct proportion to the number of DNA-1-NOP adducts formed per plasmid. At the highest level of adduct formation tested, the frequency of supF mutants was 26 times higher than the background frequency of 1.4 X 10(-4). DNA sequencing of 60 unequivocally independent mutant derived from 1-NOP-treated plasmids indicated that 80% contained a single base substitution, 5% had two base substitutions, 4% had small insertions or deletions (1 or 2 base pairs), and 11% showed a deletion or insertion of 4 or more base pairs. Sequence data from 25 supF mutants derived from untreated plasmids showed that 64% contained deletions of 4 or more base pairs. The majority (83%) of the base substitution in mutants from 1-NOP-treated plasmids were transversions, with 73% of these being G . C --> T . A. This is very similar to what we found previously in this system, using BPDE, but each carcinogen produced its own spectrum of mutations. Of the five hot spots for base substitution mutations produced in the supF gene with 1-NOP, two were the same as seen with BPDE-treated plasmids. However, the three other hot spots were cold spots for BPDE-treated plasmids. Conversely, four of the other five hot spots seen with BPDE-treated plasmids were cold spots for 1-NOP-treated plasmids. Comparison of the two carcinogens for the frequency of supF mutants induced per DNA adduct showed that 1-NOP-induced adducts were 3.8 times less than BPDE adducts. However, the 293 cell excised 1-NOP-induced adducts faster than BPDE adducts.     

Truncated variants of apolipoprotein B cause hypobetalipoproteinaemia.

Familial hypobetalipoproteinaemia is a rare autosomal dominant disorder in which levels of apo-B-containing plasma lipoproteins are approximately half-normal in heterozygotes and virtually absent in homozygotes. Here we describe mutations of the apo-B gene that cause two different truncated variants of apo-B in unrelated individuals with hypobetalipoproteinaemia. One variant, apo-B(His1795----Met-Trp-Leu-Val-Thr-Term) is predicted to be 1799 amino acids long and arises from deletion of a single nucleotide (G) from leucine codon 1794. This protein was found at low levels in very low density and low density lipoprotein fractions in the blood. The second, shorter variant, apo-B(Arg1306----Term), is caused by mutation of a CpG dinucleotide in arginine codon 1306 converting it to a stop codon and predicting a protein of 1305 residues. The product of this allele could not be detected in the circulation. The differences in size and behaviour of these two variants compared to apo-B100 or apo-B48 point to domains that may be important for the assembly, secretion or stability of apo-B-containing lipoproteins.     

Localisation of the endpoints of deletions in the 5'' region of the Duchenne gene using a sequence isolated by chromosome jumping.

We have used chromosome jumping technology to move from within a large intron sequence in the Duchenne muscular dystrophy (DMD) gene to a region adjacent to exons of the gene. The single copy jump clone, HH1, was used to characterise deletions in patients previously shown to be deleted for DNA markers in the 5' end of the gene. 12 out of 15 such patients have breakpoints which lie between HH1 and the genomic locus J-47. Thus the vast majority of the deletions in these patients have proximal breakpoints in a similar region distal to the 5' end of the gene. HH1 was mapped with respect to the X;1 translocation in a DMD female and was shown to lie at least 80 kb from the starting point of the chromosome jump, HIP25.