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E H Chang

Publications and source records attributed to E H Chang.

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

Altered messenger RNA and unique mutational profiles of p53 and Rb in human esophageal carcinomas.

Seventy-nine esophageal carcinoma patients were studied for genetic abnormalities in the p53 and Rb tumor suppressor genes. Single-strand conformation polymorphism analysis and DNA sequencing were used to detect p53 point mutations, Northern blotting was used to examine abnormal expression of p53 and Rb, and polymerase chain reaction and Southern blotting were used to analyze allelic loss. Twenty-five cases were analyzed by DNA sequencing to detect mutations in p53. Fourteen samples contained mutations within exons 5 through 9 of p53; seven had missense mutations giving rise to single amino acid substitutions. The remaining seven (50%) contained nonsense mutations leading to premature termination, five due to single base pair substitutions, and two that were the result of frameshift mutations. In other human tumors, p53 mutations are predominantly missense mutations, but our data as well as those from other groups show that nonsense mutations are common in human esophageal cancer. All but one of the constitutionally heterozygous samples containing mutations also manifested loss of the normal p53 allele; the one exception without allelic loss contained a silent mutation, which should not have had any affect on the p53 protein product. In addition, Northern blotting analysis revealed abnormalities (altered transcript size or mRNA levels) in 5 of 7 cases involving p53 and in 2 of 7 cases analyzed for Rb. Thirty-four cases were informative for allelic loss studies of both p53 and Rb; of these, 25 (74%) lost heterozygosity of p53, Rb, or both. When point mutations and mRNA expression abnormalities were also considered, 33 of 45 (73%) tumors informative for allelic loss assays of both genes as well as for mRNA or point mutation studies showed one or more abnormalities in p53 or Rb. Our results strongly suggest that a unique profile of molecular alterations involving p53 and Rb characterizes human esophageal cancer and that these specific genetic lesions are important in the development and/or progression of most human esophageal carcinomas.

Adenocarcinoma↗
PubMed1993

Expression of the c-erbB-2 (HER-2/neu) oncoprotein in human prostatic carcinoma.

The objective of this study was to determine the expression of the c-erB-2 oncoprotein via immunohistochemistry of archival clinically localized human prostate cancers and to compare these results to known clinical prognostic factors. In addition, positive staining cases were subjected to differential polymerase chain reaction to assess for c-erbB-2 gene amplification. Immunohistochemical staining with a polyclonal antibody (pAb 1) was performed on archival radical prostatectomy specimens. To standardize the staining, positive and negative control material was generated using c-erbB-2 transfected NIH3T3 cells grown on agar plugs, formalin fixed, paraffin embedded and processed on glass slides for immunohistochemistry. Definite positive membranous staining was detected in 18 of 53 neoplastic cases (34%). In addition, 9 cases of benign prostatic hyperplasia were stained without evidence of c-erbB-2 expression detected. Either focal or diffuse membranous staining was identified in 6 of 27 (22%) well, 8 of 20 (40%) moderately and 4 of 6 (66%) poorly differentiated tumors (p = 0.03, chi-square test for trend). Positive staining occurred in 6 of 18 patients (33%) with pathological stage B and 12 of 33 (36%) with pathological stage C disease. At a mean of 36 months, complete followup was available for 16 of the 18 positive cases and 30 of the 35 negative cases. For stage B 1 of 6 positive (16.7%) versus 1 of 12 negative (8%) staining cases showed progression (p = 1.0). For stage C 7 of 12 positive (58.3%) versus 9 of 21 negative (42.9%) cases showed progression (p = 0.48). Deoxyribonucleic acid was extracted from the exact same archival paraffin blocks for the c-erbB-2 protein positive cases and subjected to differential polymerase chain reaction analysis, which revealed no c-erbB-2 gene amplification. This study demonstrates that approximately a third of all clinically localized prostate cancers express the c-erbB-2 oncoprotein via immunohistochemistry using pAb-1 on archival material, c-erbB-2 oncoprotein expression does not appear to be a prognostic marker for prostate cancer although our results are preliminary and, although oncoprotein expression was detected, no positive case demonstrated deoxyribonucleic acid amplification.

ErbB Receptors↗
PubMed1993

Absent ras gene mutations in human adrenal cortical neoplasms and pheochromocytomas.

A variety of human tumors have been studied for ras mutations to date. However, little is known about the prevalence and significance of ras gene activation in adrenal neoplasms. Recently, a study of 10 primary human pheochromocytomas found no evidence for ras mutations. To our knowledge no survey of ras mutations in adrenocortical neoplasms has been reported. Therefore, we analyzed deoxyribonucleic acid (DNA) from 17 archival tumors (8 adrenocortical carcinomas, 6 pheochromocytomas, 2 adrenal adenomas, 1 aldosteronoma, 2 fresh pheochromocytomas and 1 fresh benign adrenal gland) for activating mutations at the 12, 13 and 61 codons of N-ras, H-ras and K-ras. DNA was extracted from archival tissues using 3 different methods: a simplified boiling method, a proteinase-K-phenol chloroform extraction and a novel heat-stable protease Thermus rt41A technique. The boiling and heat-stable protease methods provided for more consistent polymerase chain reaction amplifications than the more laborious phenol chloroform method. This heat-stable protease Thermus rt41A method had not been reported previously for use in archival DNA extraction. Polymerase chain reaction amplified the ras gene regions of interest, and mutations were screened by mutation-specific oligonucleotide probe hybridization of Southern and slot blots. Polymerase chain reaction-generated mutation-specific positive and negative controls were used in the hybridization protocol. With these controlled conditions no definite mutations were detected at codons 12, 13 or 61 of N, H or K-ras. Ras activation via point mutations at these codons rarely, if ever, occurs in adrenal neoplasms.

Adrenal Cortex Neoplasms↗
PubMed1993

Cytogenetic response to G2-phase X irradiation in relation to DNA repair and radiosensitivity in a cancer-prone family with Li-Fraumeni syndrome.

Noncancerous skin fibroblasts from six family members with Li-Fraumeni syndrome, five with cancer of diverse tissue origin and one with a premalignant neoplasm, showed a high frequency of chromatid aberrations, 94 to 119 breaks and 58 to 95 gaps per 100 metaphase cells arrested with colcemid 0.5 to 1.5 h after X irradiation (1.75 x 10(-2) C/kg). This response results from deficient repair of the radiation-induced DNA damage. In contrast, skin fibroblasts from two unrelated normal controls and a spouse showed 19 breaks and 17 to 19 gaps per 100 cells. Whereas all six members of the cancer-prone family had a radioresistant phenotype, only four had an inherited p53 mutation. Fibroblasts from a radioresistant family member showed the same extent of chromatid damage directly (0 to 0.5 h) after G2-phase X irradiation as those from the radiosensitive control spouse. We conclude, therefore, that radiosensitivity, as determined by cell killing in asynchronous populations of skin fibroblasts, is unrelated to chromosomal sensitivity to G2-phase X irradiation. However, the persistence of a high frequency of chromatid breaks and gaps at 0.5 to 1.5 h after G2-phase X irradiation, a manifestation of deficient DNA repair, is associated with proneness to cancer in this family.

Cells, Cultured↗
PubMed1993

Several mutant p53 proteins detected in cancer-prone families with Li-Fraumeni syndrome exhibit transdominant effects on the biochemical properties of the wild-type p53.

The identification of germ-line mutations in the p53 gene has provided a situation where comparable amounts of wild-type and mutant p53 co-exist in constitutional cells of certain individuals who are cancer-prone. Here we report the biochemical characteristics of several Li-Fraumeni syndrome associated mutant p53 proteins in order to assess the influence of germ-line mutant p53 on the functions of the wild-type p53. Unlike 248W mutant p53 protein, which was previously shown to have no effect on the wild-type p53 conformation (Milner & Medcalf, 1991; Cell 65, 765-774), germ-line associated mutant p53 proteins with residue 133T, 245D or 258K, converted the wild-type p53 conformation into the mutant conformation. Furthermore, lysates containing cotranslated wild-type p53 and these mutant p53 proteins were significantly impaired for DNA and SV40 large T antigen binding. These observations suggest that at least some germ-line p53 mutants might exhibit dominant effects on wild-type p53 functions and, like other mutant p53 proteins, the phenotype of germ-line mt p53 proteins might be variable depending on the particular mutation.

Base Sequence↗
PubMed1993

Oncogene- transformed NIH 3T3 cells display radiation resistance levels indicative of a signal transduction pathway leading to the radiation-resistant phenotype.

Oncogenes and their normal counterparts, proto-oncogenes, are functionally important cellular genes which interact with one another as components of signal transduction pathways leading to cell growth and differentiation. Numerous reports in the literature have also begun to link these genes to the phenomenon of cellular radiation resistance. In this report we examine the radiation resistance level of NIH 3T3 cells transformed by various oncogenes in an attempt to define the intracellular pathway to the radiation-resistant phenotype. The results demonstrate that an analogous signaling pathway is apparently involved in acquisition of radiation resistance. Serine/threonine protein kinase oncogenes such as raf, mos, and PKC play a central role in the pathway. Moreover, specific oncogenes upstream (sis, HER-2, met, trk, and ras) and downstream (ets and myc) of these important signaling mediators can also influence the radiation resistance level of the cells.

3T3 Cells↗
PubMed1992

Detection of RAS mutations in archival testicular germ cell tumors by polymerase chain reaction and oligonucleotide hybridization.

Preliminary studies of RAS mutational activation in human testicular germ cell neoplasms have yielded conflicting results. Whereas two studies of clinical material revealed a significant incidence of N- and KRAS mutations, two studies of a variety of germ cell lines failed to document RAS mutations. To clarify the incidence of RAS mutations in these tumors, we studied archival paraffin-embedded, formalin-fixed orchiectomy specimens from 25 nonseminomas (NSGCT), 18 seminomas (SEM), and one Leydig cell tumor. For 14 of the 44 neoplasms, DNA was also available from nonmalignant testis adjacent to the tumor. Six age-matched patients had testes removed because of nonmalignant disease and were studied as controls. Polymerase chain reaction (PCR) amplified the K-, N-, and HRAS 12, 13, and 61 codons of these specimens, and mutations were detected with mutation-specific oligonucleotide probe hybridization of Southern and slot blots. Four mutations were found in KRAS 12 (4/44;[9.1%]). One seminoma [1/18(5.6%)] contained the mutation GGT(GLY)----CGT(ARG), and three NSGCT [3/25(12%)] were found to have GGT(GLY)----GAT(ASP) mutations. One of the NSGCT mutations was detected in adjacent nonmalignant tissue, but the corresponding tumor did not contain any detectable mutation. No mutations were detected at KRAS 13 or 61, in NRAS or HRAS 12, 13, or 61, or in the control normal testes. PCR, slot blots, and hybridizations were performed twice by two separate investigators for confirmation of results. PCR-generated mutation-specific positive controls were created for all possible RAS mutations, and these along with wild-type DNA controls were integral to interpretation of the oligonucleotide mismatch hybridization assay. By using positive and negative controls, we have detected a relatively low incidence of RAS mutations in archival human testicular germ cell tumors.

Blotting, Southern↗
PubMed1992

Infrequent RAS oncogene mutations in human prostate cancer.

The RAS gene family includes three functional genes, H-RAS, K-RAS, and N-RAS, which have been most widely studied in human tumors. Point mutations most commonly occurring at codons 12, 13, or 61 of these genes allow the RAS protooncogene to be converted to a RAS oncogene. A variety of human tumors have been studied for RAS mutations to date, however, conflicting data has been reported regarding prostate cancer. Cell line studies and two American studies of clinical material have found a low incidence of RAS mutation in prostate cancer. The few mutations found were predominantly in the H-RAS gene. Conversely, a recent study of Japanese occult autopsy specimens found an approximate 25% incidence of K-RAS mutations. In this current study, DNA was extracted from 24 archival paraffin-embedded, formalin-fixed radical prostatectomy specimens. Twenty-one of the 24 cases had pathologic stage C disease, and paraffin blocks were selected having the most concentrated area of neoplasm. Twelve, seven, and five cases demonstrated moderate, well and poorly differentiated histologic grade respectively. Polymerase chain reaction (PCR) was used to amplify the K-RAS, N-RAS, and H-RAS 12, 13, 61 codons of these specimens and mutations were detected with mutation-specific oligonucleotide probe hybridization of southern and slot blots. No definite point mutations were detected. PCR's and hybridizations were performed three separate times by three investigators to confirm these results. PCR-generated mutation-specific positive controls and known negative controls were used and found to be important to interpret oligonucleotide hybridization assays. RAS gene mutations appear to be infrequent in clinical prostate carcinomas in American males.

Amino Acid Sequence↗
PubMed1992

The status of the p53 gene in human papilloma virus positive or negative cervical carcinoma cell lines.

We have analyzed p53 gene alterations in five cervical cancer derived cell lines. Two of the five cervical cancer cell lines, HTB31 (C-33A) and 32 (HT-3), harbored missense mutations in codons 273 and 245 respectively, whereas the other three tumor cell lines, HTB33 (ME180), 34 (MS751) and 35 (SIHA), did not reveal any mutation in the p53 coding sequence spanning codons 126-307. Although all the tumor cell lines express comparable levels of p53 RNA, only HTB31 and HTB32 contain high or detectable levels respectively of p53 protein. The other three tumor cell lines, where neither p53 mutation nor the expression of p53 protein could be detected, were found to harbor human papilloma virus (HPV) 16 or 18. The inactivation of the wild-type p53 function resulting from a missense mutation, or the lack of detectable wild-type p53 protein due to the translational/post-translational deregulation of p53 protein levels may be the contributing factor in the tumorigenicity of these five cases of cervical cancer. The lack of detectable p53 protein in HTB33, 34 and 35 associates with the presence of either HPV16 or -18 in these cell lines.

Amino Acid Sequence↗
PubMed1992

Detection of both mutant and wild-type p53 protein in normal skin fibroblasts and demonstration of a shared 'second hit' on p53 in diverse tumors from a cancer-prone family with Li-Fraumeni syndrome.

Germline transmission of mutant p53 gene in cancer-prone families with Li-Fraumeni syndrome has revealed a new role for p53 in the genetic predisposition to cancer. The studies reported here focus on the analysis of the expression of normal and mutant p53 RNA and protein in germline configuration and demonstrate that normal skin fibroblasts derived from members of a family with Li-Fraumeni syndrome express mutant p53Gly----Asp(245) protein and RNA at levels similar to the wild-type p53. Thus, these fibroblasts represent a unique biological system in which endogenous promoters are utilized for the expression of both mutant and normal p53. We have further extended the earlier observations on the analysis of mutant p53 with a limited number of tumors derived from individuals with Li-Fraumeni syndrome. Tumors arising from two different germ layers in four individuals in a single family clearly exhibited the loss of the wild-type allele and the retention of the mutant allele observed in the normal skin fibroblasts derived from the same individuals. These observations further support the notion that germline p53 mutation plays a key role in the tumorigenesis of individuals with Li-Fraumeni syndrome.

Alleles↗
PubMed1991

Antisense inhibition of ras p21 expression that is sensitive to a point mutation.

Many genetic disorders result from a single point mutation, and many tumor oncogenes have been found to be altered by a point mutation. The ability to inhibit selectively the expression of the mutated form of a protein without affecting its normal counterpart is central to many therapeutic strategies, since the normal protein may serve indispensable functions. Antisense oligonucleoside methylphosphonates and their psoralen derivatives directed at either normal human Ha-ras p21 or ras p21 that is mutated at a single base in codon 61 have been examined for their efficacy and specificity as inhibitors of p21 expression. Mixed cultures of cells expressing both forms of p21 were treated with the antisense oligomer complementary to the normal p21 or with the antisense oligomer complementary to the point-mutated p21. Each of the antisense oligomers specifically inhibited expression of only the form of ras p21 to which it was completely complementary and left the other form of p21 virtually unaffected.

Base Sequence↗
PubMed1991

Dynamics of differentiation in human epidermoid squamous carcinoma cells (A431) with continuous, long-term gamma-IFN treatment.

We investigated the long-term effects of continuous gamma interferon (gamma-IFN) treatment on A431, a human squamous carcinoma cell line. Cells were grown in an in vitro culture system, which over time produces cohesive cell masses ("tumoroids") exhibiting three-dimensional, histotypically differentiated structures, e.g., keratin "pearls", intercellular bridges (desmosomes), elongated flattened cells (squames) and stratification. The effects of gamma-IFN on cell growth, morphology and stage of differentiation were assessed at different treatment times by light and electron microscopy and by immunohistochemical staining using antibodies to keratins 1 and 14 and to filaggrin, markers of specific stages of keratinocyte differentiation. Our results show that A431 cells have the capacity for spontaneous differentiation, that this capacity is significantly enhanced and accelerated by gamma-IFN treatment leading to terminal differentiation and extensive cell death by 2 wk. Despite continuous exposure to IFN, a small number of viable, undifferentiated cells remain. Their proliferation, evident by 3 wk, reconstitutes the tumoroid which once again contains the full range of differentiating cell types.

Biomarkers, Tumor↗
PubMed1991

Aberrant DNA topoisomerase II activity, radioresistance and inherited susceptibility to cancer.

Inherited susceptibility to a wide variety of neoplasias (Li-Fraumeni syndrome), has been shown in studies of one cancer-prone family, to have an intriguing association with an aberrant c-raf-1 gene and inheritance of a radioresistant phenotype in their non-cancerous skin fibroblasts. This association together with observations that DNA topoisomerases, when defective, can introduce errors into DNA and that these enzymes are perturbed in vitro by serine/threonine kinases similar to raf encoded proteins, prompted investigation of DNA topoisomerase activity of the family's fibroblasts. Since radioresistance was transferred to murine cells (NIH-3T3) when the aberrant c-raf-1 gene from this family was transfected, we also examined transformants containing this and other oncogenes. V-raf/c-myc and EJ-ras transformants were examined, the former because the family's skin fibroblasts also have 3-8-fold elevated myc expression (not apparently relevant to radioresistance) and the latter because ras, like raf, conveys radioresistance. The family members' fibroblasts and the three transfected murine lines, showed a similar perturbation of a spermidine and ATP-dependent DNA catenation activity (typical of DNA topoisomerase II). There was a significant positive correlation (r = 0.93; P = 0.0026) between the degree of activation of topoisomerase II and one measure of radioresistance (the Dq value). Relaxation of DNA supercoiling (topoisomerase I activity and other DNA nicking enzymes) was not abnormal. Cytotoxicity assays and evaluation of the influence of topoisomerase II inhibitors on DNA/protein complex formation, corroborated the existence of a qualitative topoisomerase II defect in the family's cells and transfectants. Although the contention that the qualitative topoisomerase II abnormalities observed here may be associated with malfunction is highly speculative, these findings may be relevant to the mechanism of oncogenesis, not only in this family, but with raf and ras type oncogenes.

Amsacrine↗
PubMed1990

Phase I-II study of advanced head and neck squamous cell carcinoma patients treated with recombinant human interferon gamma.

The association of immunodeficiency with head and neck squamous cell carcinoma has generated the concept of supplying immunologically active agents as a means of treating these cancers. One of the most active immunologic messengers is interferon gamma, which has been observed in our laboratories to also have a direct cytotoxic effect on cultures of squamous cell carcinoma derived from the head and neck. To test the feasibility of treating patients with advanced but resectable head and neck cancer with this agent, we designed a phase I-II trial of recombinant human interferon gamma using a 24-hour infusion repeated weekly for four times. In this study, both tumor and immunologic parameters were studied before and after treatment. Eight patients were entered into the study with the highest recombinant human interferon gamma dose attempted being 0.25 mg/m2 per 24 hours. Minimal side effects were observed. Three patients had clinically measurable responses, four had stabilization of disease, and one had progression while receiving treatment. Histopathologic results of treatment were similar to in vitro observations. Necrosis, as well as differentiation of tumor cells, was observed. In some tumors there was a marked decrease in cellularity without a change in tumor volume due to increased extracellular keratin deposition. Our study indicates that evaluation of adoptive immunotherapy trials in head and neck cancer needs to include parameters other than simple tumor regression as an end point, otherwise therapeutically important lymphokine-induced changes may be missed. Further evaluation of recombinant human interferon gamma and agents that induce human interferon gamma are warranted.

Adult↗
PubMed1989

raf involvement in the simultaneous genetic transfer of the radioresistant and transforming phenotypes.

We examined a human Alu+ mouse tertiary transformant derived from a noncancerous skin fibroblast cell line which exhibits the unique characteristic of being resistant to the killing effects of ionizing radiation. This transformed cell line was found to contain activated human c-raf-1, and demonstrated an increased level of radioresistance indicating the simultaneous transfer of both the transforming and radiation-resistant phenotypes. We have also found a relationship between the presence of activated oncogenes, specifically those with serine/threonine kinase activity and the radioresistant phenotype.

Animals↗
PubMed1989

A study of the bronchial provocation test with methacholine in patients with active pulmonary tuberculosis.

Bronchial hyperreactivity is a characteristic feature of bronchial asthma. Recent respiratory infections, allergic rhinitis, atopic family history, pulmonary tuberculosis, pulmonary sarcoidosis, cystic fibrosis, and farmer's lung have also been demonstrated to have bronchial hyperreactivity to inhaled methacholine. It is not known if pulmonary tuberculosis can cause nonspecific bronchial hyperreactivity and what the mechanism would be. We therefore undertook to evaluate nonspecific bronchial hyperreactivity in active pulmonary tuberculosis using the bronchial provocation test with methacholine and we measured the total serum IgE and peripheral eosinophil count to seek some mechanisms. There were 5 patients among 18 subjects with active pulmonary tuberculosis whose response to methacholine was positive. The mean baseline FEV1 of positive responders was 71.40 +/- 17.39%, and that of negative responders was 110.18 +/- 17.65% (p less than 0.05). There were no significant differences in serum IgE and peripheral eosinophil count between positive and negative responders. We found that active pulmonary tuberculosis would increase the nonspecific bronchial response with methacholine, and the mechanism of the bronchial hyperreactivity in patients with active pulmonary tuberculosis may not be related to an immunologic mechanism but may be related to the stimulating receptors.

Acute Disease↗
PubMed1989

Sequence specific inhibition of in vitro translation of mutated or normal ras p21.

Antisense methylphosphonate-modified oligomers (ONMP) complementary to 8 nucleotides spanning the twelfth amino acid codon of human c-Ha-ras have been synthesized to explore their inhibitory effect on ras p21 translation. The ONMP Ras 0, perfectly complementary to the specific target region in normal c-Ha-ras, inhibits cell-free translation of p21 from a normal c-Ha-ras mRNA template in a dose-dependent manner. At 200uM Ras 0, p21 translation is inhibited by almost 90% in a rabbit reticulocyte lysate; at 100uM, p21 is reduced by over 60%. Ras I and Ras II contain, internally, a single and double base mismatch, respectively. The inhibitory activity of these ONMPs is reduced in proportion to the number of mismatches. When the target mRNA encodes the activated c-Ha-ras differing by a single nucleotide at the twelfth amino acid codon from normal c-Ha-ras, the magnitude of the inhibitory effect of Ras I increased significantly because Ras I is now perfectly complementary to its target mRNA. In turn, Ras 0, now only partially complementary, is considerably less effective at the same concentrations. Therefore, the inhibitory effect is exquisitely sensitive to the extent of the sequence complementarity between the antisense ONMP and the targeted mRNA.

Amino Acids↗