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Biomedical subjects

W Warren

Publications and source records attributed to W Warren.

At least 37 records · Page 2Linked to original sources
PubMed1993

Does a genotoxic carcinogen contribute to human breast cancer? The value of mutational spectra in unravelling the aetiology of cancer.

The p53 tumour suppressor gene is turning out to be a useful reporter for the stigmata of past genotoxic exposure. About half of all human cancers contain p53 mutations most of which occur in those regions (exons 5-8) of the gene that are highly conserved during evolution. Mutations are mainly of the missense type and their frequency and distribution vary among different kinds of cancer. The ability to detect all six possible base-substitution mutations in the p53 gene in human tumours makes it possible to construct mutational spectra for different cancers at a locus clearly implicated in carcinogenesis. Transitions at one particular hotspot--the CpG dinucleotide--occur frequently in many cancers and may reflect endogenous mutation. A reduction in the proportion of CpG mutations at the expense, for example, of an increase in GC to TA transversions may signal the effect of an exogenous mutagen. We exploited these features of the p53 gene to examine the evidence that a previously unsuspected genotoxic exposure may contribute to the high incidence of breast cancer in women living in rich industrialized countries. We compiled a mutational spectrum of p53 from 120 breast cancers and compared it with the spectrum from 145 colorectal cancers and 246 lung cancers. A germline p53 spectrum was constructed using data from 27 patients. Two hundred germline mutations in the haemophilia B gene served as a 'background' spectrum.(ABSTRACT TRUNCATED AT 250 WORDS)

Breast Neoplasms↗
PubMed1993

Identification and localization of bacterial endosymbionts in hydrothermal vent taxa with symbiont-specific polymerase chain reaction amplification and in situ hybridization techniques.

Invertebrates that contain endosymbiotic chemoautotrophic eubacteria are widely distributed in a variety of reducing marine habitats, including deepsea hydrothermal vents. The mechanisms of symbiont transmission in these invertebrates are not understood. To test the hypothesis that symbionts are transmitted via the eggs of hosts, we used group-specific hybridization probes complementary to 16S ribosomal RNAs (rRNAs) to look for symbionts in eggs and ovaries. 16S rRNA sequences were examined for domains unique to the symbionts of three vent animals: Calyptogena magnifica, Bathymodiolus thermophilus, and Riftia pachyptila. Three 16S rRNA-directed oligodeoxynucleotide hybridization probes (CG-1255R, RP-1243R, BT-1255R) specific for these endosymbionts were synthesized and evaluated by dot-blot hybridization. At higher stringencies, all three probes showed a high degree of specificity for their target endosymbionts rRNAs. The probes were also used as polymerase chain reaction (PCR) primers for detection of the symbiont 16S rRNA genes in genomic DNA isolated from host tissues known to contain symbionts. All three symbiont-specific probes were highly sensitive and specific as PCR primers; they successfully amplified 1 pg target DNA. However, all amplifications of extracted egg DNA from the vestimentiferan R. pachyptila with either universal eubacterial (Eub A/B) or the Riftia symbiont-specific (RP-1243R/Eub B) primer sets were unsuccessful. Nonradioactive in situ hybridizations were performed on ovarian tissue from the vestimentiferan Ridgea piscesae using RP-1243R, 3' end-labeled with digoxigenin-11-dUTP (Boehringer Mannheim). The probe was subsequently detected with an alkaline phosphatase-conjugated immunoglobulin G antibody specific for the digoxigenin moeity. The probe bound only to the tissue of R. pisceasae coincident with the known location of symbiont cells and was not detected in any region of the ovary. These data indicate that transovarial symbiont transmission in the vestimentiferans does not take place and that symbiont acquisition is probably a post-spawning event.

Animals↗
PubMed1993

Constitutional mutation in exon 8 of the p53 gene in a patient with multiple primary tumours: molecular and immunohistochemical findings.

We report a constitutional mutation of codon 273 in exon 8 of the p53 gene. The affected individual has developed multiple independent benign and malignant tumours (tricholemmoma of the scalp, multiple trichoepitheliomata of the face, osteosarcoma of the ovary, bilateral breast cancer, malignant fibrous histiocytoma of the thigh and endometrial adenocarcinoma) and belongs to a family with some, but not all, features of the Li-Fraumeni syndrome. The mutation, found in both blood lymphocyte and tumour specimens, is a cytosine to thymine transition at codon 273, resulting in an amino acid change from arginine to cysteine. The mother and sister of the index case both died of tumours at an early age. We have demonstrated that formalin-preserved material from these tumours contains the same C-->T mutation at codon 273, indicating that this mutation has probably been transmitted through the germline. All tumours from the index case, both benign and malignant, showed immunohistochemical positivity with four antibodies to the p53 protein. Positive staining was also seen in scattered nuclei of morphologically normal epidermal keratinocytes and pilosebaceous cells, but not in lymphocytes or other morphologically normal cells from the index case. However, a similar staining pattern in apparently normal tissue was also observed in 13/48 sections from other individuals with various skin conditions (melanocytic naevi, psoriasis and normal skin adjacent to malignant melanoma and fibrous histiocytomas), suggesting that this pattern of p53 staining may not be unique to individuals with constitutional p53 mutations.

Base Sequence↗
PubMed1992

Analysis of the p53 tumor-suppressor gene in hepatocellular carcinomas from Britain.

Human hepatocellular carcinomas from patients in Britain, an area of low prevalence of hepatocellular carcinoma and low dietary exposure to aflatoxin B1, were analyzed for mutations in the p53 tumor-suppressor gene. Abnormalities in the p53 gene were detected in 2 of 19 hepatocellular carcinomas by polymerase chain reaction--single-stranded conformation polymorphism. Direct sequencing of the evolutionarily conserved regions of p53 (exons 5, 6, 7 and 8), where mutations have been commonly found in a variety of tumors, confirmed that only two hepatocellular carcinomas had mutations in p53, one a 6-bp deletion of codons 158 and 159 (exon 5) and the other a G to A transition at codon 286 (exon 8). No mutations were found in any hepatocellular carcinoma in exons 6 and 7; in particular all tumors had wild-type sequence at codon 249, which has been reported to be a mutational hot spot in the p53 gene in hepatocellular carcinomas from high incidence areas such as China and southern Africa. Abnormalities in p53 expression were examined by immunohistochemistry and found in 1 of the 19 hepatocellular carcinomas. These findings show that p53 mutations are infrequently involved in the malignant transformation of hepatocytes in an area of low hepatocellular carcinoma prevalence. They support the suggestion of a possible link between dietary exposure to aflatoxin and selective G to T mutations at codon 249 of the p53 gene. Our observations also indicate that hepatitis B virus infection alone, present in six of the hepatocellular carcinomas examined, does not account for the specificity for codon 249 mutations reported from endemic areas.

Base Sequence↗
PubMed1992

Combined modality therapy for stage III non-small cell lung carcinoma: results of treatment and patterns of failure.

Patients with Stage III non-small cell lung carcinoma continue to pose a therapeutic problem with dismal cure rates. In an effort to improve on these results, 129 patients with biopsy-proven clinical Stage III non-small cell lung carcinoma from November 1982 through November 1987, were entered into two consecutive Phase II studies at Rush-Presbyterian-St. Luke's Medical Center. Treatment in the first study consisted of Cisplatin and 5-Fluorouracil infusion with concomitant split course radiation; in the second Etoposide was added. Radiation and chemotherapy were given simultaneously on days one through five of each cycle in a preoperative fashion for four cycles in patients considered eligible for surgery and in a definitive fashion for six cycles in patients considered ineligible for surgery. Radiation was given in 2 Gy fractions for a planned preoperative dose of 40 Gy and a definitive dose of 60 Gy. Surgical resection was attempted four to five weeks later in patients treated preoperatively. Thus, 83 patients were treated preoperatively and 46 definitively. Eighty-three patients (64%) had IIIA disease and IIIB disease was found in the remainder of the patients. Sixty-two patients (75%) in the eligible for surgery group had a thoracotomy after the combined treatment with a resectability rate of 97% and an operative mortality rate of 5%. There were 17 patients (27%) with no evidence of residual cancer in the resected specimen. Three-year survival for the eligible for surgery group at 40% was significantly better than 19% observed in the ineligible for surgery group (p = 0.003). Seventy-six percent of the patients with no residual cancer in the resected specimen are recurrence-free at three years compared to 34% of the patients with gross residual. A total of 81 patients have failed after their treatment; 49 (59%) in the eligible for surgery group and 32 (70%) in the ineligible for surgery group. Of all the patients who failed, local failure alone and as a component occurred in 21 (26%) and 36 (44%) patients, respectively. Failure in distant sites alone was noted in 56% of the overall failures. Severe toxicity was unusual. There were three treatment related deaths (2%). Radiation esophagitis and pneumonitis were only mild to moderate seen in less than 10% of the patients. Survival rates and patterns of failure according to the stage of the disease, histology, treatment group and pathologic response will be presented in detail.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1992

Increased expression and mutation of p53 in choroidal melanoma.

Using CM-1 antibody directed against the human p53 protein, high levels of mutant p53 protein expression were found in 12 out of 18 malignant choroidal melanomas. In contrast, we failed to observe elevated p53 expression, indicating the absence of p53 mutation in seven choroidal naevi, a potentially premalignant condition that can progress to form malignant melanoma. For two choroidal melanomas, we demonstrated that high levels of p53 protein were accompanied by exon 7 mutations. The mutations were found at codon 238, TGT-->TTT and codon 253, ACC-->AGC. These observations suggest that acquisition of abnormalities of the p53 gene may be an important step in the development of malignant melanoma.

Animals↗
PubMed1992

No evidence for germline mutations in exons 5-9 of the p53 gene in 25 breast cancer families.

Recent studies have demonstrated that families with the Li-Fraumeni syndrome carry inherited point mutations of the p53 gene. In the present study 25 families with strong histories of breast cancer were screened for the presence of such mutations. Polymerase chain reaction products of exons 5-9 of the p53 gene were examined by single-stranded conformational polymorphism analysis and, in addition, exon 7 was further screened by direct sequencing. No mutations were detected in constitutive DNA by either method. These results indicate that familial breast cancer does not usually result from germline point mutations in the p53 gene.

Breast Neoplasms↗
PubMed1992

Large-scale purification of plasmid DNA by anion-exchange high-performance liquid chromatography.

Numerous methods have previously been reported for the final steps in the large-scale purification of plasmid DNA. Although gel permeation and reverse-phase high-performance liquid chromatography have been utilized for this procedure in the past, the limited capacity of these systems often necessitated multiple rounds of chromatography, especially with the high copy number plasmids commonly in use today. In this paper, the use of the high-capacity, high-resolution Protein-Pak DEAE 8HR column is presented for the large-scale isolation of highly purified plasmid DNA from crude E. coli cell lysates. Up to 5 mg of plasmid DNA have been purified in a single 50-minute chromatography run. The purified DNA demonstrated excellent biological activity as demonstrated by restriction endonuclease digestion, E. coli transformation and DNA-mediated gene transfection of eukaryotic cells.

Biotechnology↗
PubMed1991

Pre-treatment prognostic factors in stage III non-small cell lung cancer patients receiving combined modality treatment.

Approximately one-third of non-small cell lung cancer (NSCLC) patients present with locally advanced disease. Increasing numbers of clinical trials are being conducted in this group of patients and recently a new international staging system has been introduced, resulting in the sub-division of Stage III into IIIa (potentially operable disease) and IIIb (inoperable disease). Kaplan-Meier survival analyses and Cox regression analyses were used to analyze data from 129 Stage III NSCLC patients who had been treated on two consecutive Phase II trials testing combined modality treatment. The pretreatment characteristics of these patients were: median age--59 years, males/females--87/42, caucasian/non-caucasian--111/18, squamous cell or adenocarcinoma/large cell carcinoma--57/72, previous weight loss less than or equal to 5%/greater than 5%-76/46, previous history of cardiorespiratory disease--no/yes--91/36, performance status (PS) 0-1/2-3--102/27, Stage III, 2 groups--IIIa/IIIb--83/46, Stage III, 3 groups--IIIa T3 N0/IIIa N2/IIIb--41/41/47, surgical eligibility--eligible/ineligible--83/46. Kaplan-Meier statistics revealed significantly longer survival for PS 0-1 versus 2-3 (p = .001), for eligible versus ineligible for surgery (p = .003), for Stage-IIIa versus IIIb (p = .004), and for Stage-IIIa T3N0 versus IIIa N2 versus IIIb (p = .004). The best model developed from Cox regression analyses included stage (IIIa T3 N0 vs IIIa N2 vs IIIb), PS, and sex. These observations appear to have implications for clinical research in Stage III NSCLC.

Adenocarcinoma↗
PubMed1991

Expression of p53 in premalignant and malignant squamous epithelium.

Using three antibodies (JG8, CM-1 and 1081) directed to the p53 protein, strong positivity was found in 16/47 (34.0%) of mucosal squamous cell carcinomas of the head and neck and in two squamous carcinoma cell lines (LICR-LON- HN5 and HN6Rr). The presence of the mutant p53 was confirmed in the cell lines as substitutions in exon 7 (codon 238, TGT greater than AGT) and exon 5 (codon 152, CCG greater than CTG) respectively. Positive staining was seen only in the undifferentiated cells and progressively lost as the cells keratinized, both in the tumour specimens and in the cell lines. Similar results were seen in areas of dysplasia, well removed from the site of the primary tumour. Staining of epidermal lesions showed positivity in 2/12 (16.6%) cases of Bowen's disease, 0/12 (0.0%) cases of solar keratosis, 0/10 (0.0%) basal cell carcinomas and in 3/20 (15.0%) squamous cell carcinomas. These results are discussed in relation to the multifocal origin of squamous cell carcinomas, the role of p53 mutations in squamous cell carcinomas from different sites and the significance of the 'basal' distribution of p53 as a normal growth regulator. The possible significance of the distribution of p53 in squamous epithelium as it relates to papilloma virus infection is also considered.

Amino Acid Sequence↗
PubMed1991

Rapid analysis and quantitation of PCR products by high-performance liquid chromatography.

HPLC utilizing a high efficiency anion-exchange column provides a rapid and easily automated technique for the qualitative and quantitative analysis of subnanogram to microgram amounts of DNA fragments generated by the PCR. The accuracy, precision and linearity of this method exceed those attainable with electrophoretic techniques. In addition, because of the nondestructive nature of HPLC, the desired PCR amplification product can be purified for subsequent utilization. Thus, liquid chromatography extends the utility of the PCR technique to those applications requiring precise quantitation.

Base Sequence↗
PubMed1991

HPLC purification of polymerase chain reaction products for direct sequencing.

Same day PCR amplification and sequencing is desired in situations where one needs to sequence a number of PCR products. The rapid, high-yield purification of PCR products via the use of high performance, anion-exchange chromatography yields sequencing results comparable to those obtained from techniques requiring subcloning of the PCR product. This can be achieved by standard dideoxynucleotide sequencing technology without the need to prepare prelabeled primers and additional internal primers or to gel purify the PCR product. In addition, this chromatographic technique offers the potential of isolating several PCR products from the same amplification mixture.

Base Sequence↗
PubMed1990

New family of high-resolution ion exchangers for protein and nucleic acid purifications from laboratory to process scales.

A new family of polymer-based ion exchangers was tested for the purification of acidic and basic proteins on both the analytical and preparative scales. Protein-Pak HR series packings are available as strong cation (SP) and weak anion (DEAE) exchangers, allowing the development of a purification method regardless of the isoelectric point of protein. Three particle sizes, 8, 15 and 40 microns, are offered in scalable Advanced Purification (AP) glass columns or as bulk packings. The lower back pressures of the 15- and 40-microns packings compared to that of the 8-microns material allow rapid throughput of large volumes without exceeding the pressure limitations of the resin or the column. The capacity of the AP1 (100 mm x 10 mm) glass columns, containing these ion-exchange packings, is comparable to other ion-exchange columns. The resolution of mouse serum, plasmids, and a standard protein mixture was demonstrated and compared with the results obtained with other resin-based ion exchangers of similar particle size. Proteins were purified without significant loss of biological activity or mass.

Alkanesulfonates↗
PubMed1990

Combined modality therapy for locally advanced non-small cell lung carcinoma.

Multi-modality treatment consisting of cisplatin, VP-16, and 5-fluorouracil chemotherapy given concomitantly with external beam radiation was used to treat 64 patients with locally advanced Stage III non-small cell lung carcinoma. This regimen was used in a preoperative fashion for four cycles in patients considered surgically resectable and with curative intent for six cycles in the remainder of patients. The clinical response rate for the entire group was 84% and the overall local control rate was 74%. The median survival was 13 months with a median follow-up for live patients of 19 months. The actuarial 3-year survival and disease-free survival rates were 30% and 23%, respectively. Histologic complete response was 39% and appeared to predict for survival. The 3-year actuarial survival and disease-free survival rates for 23 resected patients were 69% and 45%, respectively, with the complete histologic responders having a disease-free survival of 78%. The pattern of first recurrence did not appear to differ by histology or presence of lymph nodes in this subset of patients. The actuarial 3-year survival and disease-free survival rates for inoperable patients receiving six cycles of treatment were 18% and 23%, respectively. The local control was 67% with the majority of these patients having Stage IIIB disease. The Mountain International staging system appeared to predict for operability, local recurrence, and survival. This concomitant treatment regimen is feasible, with the major toxicities being leukopenia, nausea, and vomiting.

Adult↗
PubMed1990

Chemical induction of thymomas in AKR mice: interaction of chemical carcinogens and endogenous murine leukemia viruses. Comparison of N-methyl-N-nitrosourea and methyl methanesulphonate.

The time course of development of thymic lymphoma, which occurs spontaneously in mice of the AKR strain, is accelerated by the methylating agents N-methyl-N-nitrosourea (MNU) and methyl methanesulphonate (MMS). Since MNU is a potent mutagen inducing G----A transition mutations and MMS a relatively weak mutagen, it was of interest to examine the genetic alterations associated with each class of the chemically induced tumors and to compare these alterations with those found in the spontaneous tumors. The same spectrum of genetic alterations was found for MMS-induced and spontaneous thymomas. Both showed rearrangements of c-myc and Pim-1 genes that appeared to result from integration of recombinant mink cytopathic focus-forming (MCF) proviruses but failed to reveal evidence for activation of ras oncogenes, either by DNA transfection experiments or by hybridization of DNA to specific oligonucleotide probes. Some alteration in c-myc and Pim-1 genes were also found in MNU-induced tumors, but, mainly, these involved integration of ecotropic-like rather than recombinant MCF viruses. Furthermore, MNU-induced tumors frequently (in 24% of thymomas) contained G----A transition mutations, activating the Ki-ras oncogene at codon 12 position 2. Another feature that distinguishes the MNU-induced tumors from those occurring in untreated and MMS-treated mice was the consistently high level of c-myc mRNA that occurred in the absence of c-myc gene rearrangement. Taken together, the data indicate that the mechanisms of development of tumors following treatment with MNU and MMS are distinct, and that the effect of MMS is probably to speed up the process of viral leukemogenesis.

Animals↗
PubMed1990

Mutation of the p53 gene in human soft tissue sarcomas: association with abnormalities of the RB1 gene.

Soft tissue sarcomas have been examined for alterations in the p53 gene. In six sarcomas, loss or rearrangement of both alleles of this gene was detected while in a further seven sarcomas, point mutation or absence of transcription of the p53 gene was observed. Abnormalities of the p53 gene were found in several classes of soft tissue sarcoma, including leiomyosarcomas, rhabdomyosarcomas and malignant fibrous histiocytomas. Our studies also show that abnormalities of the RB1 suppressor gene and of the p53 gene frequently occur together. These results are consistent with the idea that the p53 gene is a tumour suppressor gene and indicate that coincident inactivation of more than one tumour suppressor gene may, in some cases, be required for tumour development.

Base Sequence↗