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J D Minna

Publications and source records attributed to J D Minna.

At least 145 records · Page 8Linked to original sources
PubMed1992

p53: a transdominant regulator of transcription whose function is ablated by mutations occurring in human cancer.

Gal4-p53 fusion constructs demonstrate that wild type p53 is a potent transactivator in human lung cancer cells with the transactivation domain for p53 residing in amino acids 1-42. Strikingly, a variety of lung cancer derived p53 mutations occurring outside this domain disrupt this activity. Temperature sensitive conformational shifts of p53 mutant proteins to the wild type form exist and, with a temperature downshift, several mutants become transcriptionally active. Wild type p53 protein is known to form oligomers with mutant p53 and cotransfection of wild type and mutant genes shows that p53 acts in a transdominant manner that is independent of the DNA binding specificity. Transcription is either increased or decreased depending on whether the wild type is more or less abundant than the mutant form. Finally, lung cancers differ in their ability to support the transactivation related functions, providing evidence of other abnormalities of the p53 system in human cancer.

Amino Acid Sequence↗
PubMed1992

Biologically active MK-801 and SKF-10,047 binding sites distinct from those in rat brain are expressed on human lung cancer cells.

We have shown previously that cultured human lung cancer cells of different histologic types express multiple opioid receptors that can regulate their growth. In this report, we show that these cells also express specific, saturable, and high-affinity binding sites (Kd approximately 1 nM) for the non-opioid phencyclidine (PCP), [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,b]cyclohepten-5,10-imine hydrogen maleate] (MK-801) and sigma N-allylnormetazocine (SKF-10,047) receptor ligands. Characterization of these binding sites showed them to be protein in nature and sensitive to the guanine nucleotide GTP. Pharmacological studies showed that (+) MK-801 and (+) SKF-10,047 competed with each other for their binding sites and also for the methadone binding site present in these cells. However, the mu and delta opioid ligands did not compete for (+) MK-801 and (+) SKF-10,047 binding sites. In addition, these binding sites on lung cancer cells appear to be distinct from the N-methyl D-aspartate/PCP receptor ionophore complex reported to be present in rat brain. MK-801 and SKF-10,047, at nM concentrations, were found to inhibit the growth of these cells in culture within a few hours of exposure, and this effect was irreversible after 24 h. The growth effects of these ligands could not be reversed by the opioid antagonist naloxone, suggesting involvement of nonopioid type receptors in the actions of these ligands. The abundant expression of biologically active MK-801 and SKF-10 047 binding sites in these cell lines, distinct from those in rat brain, suggests that these cell lines may prove to be a valuable source for further characterization and purification of these binding sites.

Animals↗
PubMed1992

Interaction of the human papillomavirus type 16 E6 oncoprotein with wild-type and mutant human p53 proteins.

The E6 oncoproteins encoded by the cancer-associated human papillomaviruses (HPVs) can associate with and promote the degradation of wild-type p53 in vitro. To gain further insight into this process, the ability of HPV-16 E6 to complex with and promote the degradation of mutant forms of p53 was studied. A correlation between binding and the targeted degradation of p53 was established. Mutant p53 proteins that bound HPV-16 E6 were targeted for degradation, whereas those that did not complex HPV-16 E6 were not degraded. Since the HPV-16 E6-promoted degradation involves the ubiquitin-dependent proteolysis pathway, specific mutations were made in the amino terminus of p53 to examine whether the E6 targeted degradation involved the N-end rule pathway. No requirement for destabilizing amino acids at the N terminus of p53 was found, nor was evidence found that HPV-16 E6 could provide this determinant in trans, indicating that the N-terminal rule pathway is not involved in the E6-promoted degradation of p53.

Amino Acid Sequence↗
PubMed1992

p53 gene mutations in non-small-cell lung cancer cell lines and their correlation with the presence of ras mutations and clinical features.

We screened 77 non-small-cell lung cancer (NSCLC) cell lines for mutations of the p53 gene using a single-strand conformation polymorphism (SSCP) assay. We found that 57 cell lines (74%) had mutations of the p53 gene. Three cell lines had a deletion of the p53 gene. Of the remaining 54 cell lines, 49 cell lines were sequenced and 52 mutations were confirmed. In contrast to previously published p53 mutations in other human tumors, the p53 gene mutations in NSCLC were diverse with regard to the location and nature of the mutations. The region corresponding to codons 144-166, which is outside the evolutionarily conserved regions, was a frequent site of p53 gene mutations in NSCLC. The presence of a p53 gene mutation was not associated with age, sex, histological types, culture site, treatment intent, presence of prior cytotoxic treatment, neuroendocrine differentiation, median culture time or patient survival. The prevalence of p53 mutations in cell lines with ras mutations did not differ from that in cell lines without ras mutations. However, p53 gene mutations in NSCLC cell lines with ras mutations tended to cluster in exon 8, suggesting the presence of a functional domain of the p53 gene relating to interaction with the ras gene. We conclude that p53 and ras mutations are frequent and apparently independent genetic alterations which play different roles in the pathogenesis, progression and prognosis of NSCLC.

Amino Acid Sequence↗
PubMed1992

Expression of surfactant associated protein-A and Clara cell 10 kilodalton mRNA in neoplastic and non-neoplastic human lung tissue as detected by in situ hybridization.

Two markers for the progenitor cells of peripheral airways and their tumors are the 10 kilodalton (kd) Clara cell protein and the major surfactant associated protein-A (SP-A). We used the RNA-RNA in situ hybridization technique to study expression of the genes encoding these proteins at the cellular level in 19 pairs of non-neoplastic and neoplastic tissues from resected human lungs. Our results show that in non-neoplastic lung tissue, the Clara 10 kd protein gene was expressed in nonciliated cells of both bronchial and bronchiolar epithelium, indicating that, in contrast to previous assumptions, cells with Clara cell-like differentiation in humans may not be restricted to bronchiolar cells. The incidence of Clara 10 kd protein gene expression, as detected in lung carcinomas (1 out of 19 cases positive) was less than expected based on previous ultrastructural reports. The SP-A gene was strongly expressed in normal alveolar type II cells in non-neoplastic lung and, at higher levels, in hyperplastic cells. In addition, SP-A mRNA expression was observed in scattered bronchial and bronchiolar epithelial cells in 40% of the airways examined. Five out of 17 lung tumors, all of which were adenocarcinomas, were positive for SP-A expression, albeit generally less intense than type II cells. This expression was seen in carcinomas with papillolepidic as well as solid and glandular growth patterns. Our findings provide new insights into the peripheral airway cell differentiation.

Carcinoma, Non-Small-Cell Lung↗
PubMed1992

[Mutations of ras and p53 genes in human non-small cell lung cancer cell lines and their clinical significance].

We examined 77 non-small cell lung cancer (NSCLC) cell lines for mutations of 3 ras genes and p53 gene, and ras mutations were detected by designed RFLP assay generated by mismatched primers during the PCR step and p53 gene mutations were detected using SSCP analysis. The incidence of ras mutations were 27/77 (35%) while that of p53 gene mutations were 57/77 (74%). The incidence of ras mutations in cell lines with p53 mutations were not different from that without p53 mutations, suggesting that they occurred independently. Neither ras nor p53 mutations correlated with histologic subtype, disease extent, in vitro culture time nor prior therapy status. The patients whose cell lines had any ras mutations survived for shorter period of time not only among the patients who were treated with curative intent but among those treated with palliative treatment. The Cox proportional hazards model predicted the higher risk for patients with ras mutations but not those with p53 mutations. We conclude that ras and p53 mutations are frequent, apparently independent genetic alterations which play different roles in NSCLC and that this information should be utilized in surgical oncology in the near future.

Carcinoma, Non-Small-Cell Lung↗
PubMed1992

Expression of mutant p53 proteins in lung cancer correlates with the class of p53 gene mutation.

We investigated the immunocytochemical staining and immunoblotting characteristics of 33 different p53 mutant proteins identified in lung cancer cell lines (18 small-cell lung cancer and 15 non-small-cell lung cancer) using monoclonal antibodies pAbs 240, 421 and 1801. The p53 mutants studied were representative of those found in lung cancer and included three deletions, four nonsense, seven splicing and 19 missense lesions. Control cell lines included six B-lymphoblastoid cell lines and two lung cancer cell lines without p53 mutations. Immunocytochemistry demonstrated 16 cell lines (48%) with definite overexpression of p53 protein (the high-expresser group of mutants), while in the remainder of cases either no p53 expression or low levels of p53 protein expression were found (the low-expresser group of mutants). The type of p53 mutation correlated with the expresser group. High expressers all had p53 missense mutations in exons 5-8, and immunocytochemistry identified 16/17 (94%) of these mutants. Several classes of p53 mutations occur in the low-expresser groups: deletions, splicing mutants, nonsense mutants and missense mutations outside of exons 5-8 all resulted in very low or undetectable levels of p53 protein. We conclude that there are low- and high-expression groups of p53 mutants in lung cancer and that the detection of protein expression in tumor cells by immunocytochemistry and immunoblotting is dependent upon the type of mutation of the p53 tumor-suppressor gene.

Blotting, Western↗
PubMed1991

ras gene mutations in non-small cell lung cancers are associated with shortened survival irrespective of treatment intent.

We analyzed 66 non-small cell lung cancer cell lines for mutations at codons 12, 13, and 61 of all three ras genes and correlated the findings with patient survival. We used designed restriction fragment-length polymorphisms to detect mutations after amplification of ras-specific sequences by the polymerase chain reaction. We found 19 mutations of ras genes (29%), and 11 of these 19 (58%) were at codon 12 of the K-ras gene. By univariate analysis, the presence of any ras mutation in cell lines from patients who received curative intent treatment was associated with a shorter survival (P2 = 0.002). For patients who received only palliative treatment, detection of K-ras mutations at codon 12 was associated with a shortened survival (P2 = 0.0103), but this analysis was not statistically significant for the group with any ras mutation (P2 = 0.093). The Cox proportional hazards model also predicted a higher risk for patients with any type of ras mutations. We conclude that ras mutations, present in a subset of non-small cell lung cancers, are independently associated with the shortened survival of patients, irrespective of treatment intent.

Analysis of Variance↗
PubMed1991

Occurrence of p53 gene abnormalities in gastric carcinoma tumors and cell lines.

We explored the state of the p53 gene in gastric cancer. Using one or more methods, we examined 15 specimens from primary carcinomas (14 tumors, one cell line), five cell lines derived from metastases, and seven paired samples of nonmalignant gastric mucosa. Sequence analyses of complementary DNA containing the entire p53 gene open reading frame demonstrated abnormalities in one of five samples from primary tumors and in all five samples from metastases. The single cell line derived from a primary carcinoma had no abnormality of the gene. The six abnormalities included four point mutations, one base-pair deletion resulting in a frame shift, and a 24 base-pair deletion caused by an intronic point mutation (as determined by sequence analysis of genomic DNA). Four of the six mutations mapped to regions highly conserved among species or involved in simian virus 40 T-antigen binding. Restriction fragment length polymorphism studies confirmed that chromosome 17p allelic deletions occur only in a minority of primary tumors, but that they may occur more frequently in metastases. Northern blotting and ribonuclease protection assays detected only a fraction of the p53 gene abnormalities detected by sequencing. Our findings indicate that mutations of the p53 gene are relatively rare in primary gastric tumors but appear to be relatively frequent in cell lines derived from metastatic lesions. Our results may help in understanding the molecular events associated with progression and metastasis in gastric carcinoma.

Amino Acid Sequence↗
PubMed1991

Nonrandom structural and numerical chromosome changes in non-small-cell lung cancer.

Cytogenetic studies were performed on 27 tumor cell lines (most of which were derived from metastatic lesions) and four fresh malignant pleural and pericardial effusions from 30 patients with non-small-cell lung cancer (non-SCLC). Many clonal structural (deletions and nonreciprocal translocations) and numerical abnormalities were found in each specimen. Statistical analysis revealed these changes were nonrandomly distributed among the chromosomes. A statistically significant number of chromosomal breakpoints were seen in regions 1q1, 1q3, 3p1, 3p2, 3q1, 3q2, 7q1, 13p1, 14p1, 15p1, and 17q1 when the regions were compared to the total haploid complement. In addition, when a given region was compared to other regions within the same chromosome, statistically significant numbers of breakpoints were noted for regions 1q3, 5q1, 7q1, 13p1, 14p1, 15p1, 16q2, 17q1, and 21p1. Specific chromosome bands showing the most frequent involvement in structural abnormalities were (in descending order) 3p14.2, 3q21, 19q13, 11p15, 1q11, 7q11, 1q21, 3p23, and 3p21. The breakpoints indicate areas to look for new dominant oncogenes activated by translocations, while the areas of deletions and loss of material by nonreciprocal translocations highlight areas to search for recessive oncogenes. These cytogenetic studies represent strong evidence that multiple genetic lesions are associated with the development of metastatic lung cancer, and provide a roadmap to search for new genes involved in the pathogenesis of lung cancer.

Carcinoma, Non-Small-Cell Lung↗
PubMed1991

Mutations of ras genes distinguish a subset of non-small-cell lung cancer cell lines from small-cell lung cancer cell lines.

We screened a panel of 103 human lung cancer cell lines for the presence of point mutations at codons 12, 13 or 61 of the human K-, H- and N-ras genes, using restriction fragment length polymorphisms (RFLP), created through mismatched primers during polymerase chain reaction (PCR) of genomic DNA. We found ras mutations in 22/61 (36%) non-small-cell lung cancer (NSCLC) cell lines, predominantly in K-ras codon 12. Identical mutations were present in uncultured tumor materials corresponding to 11 cell lines containing mutated ras genes. ras mutations were found not only in adenocarcinoma cell lines (9/32, 28%), but also in cell lines derived from other types of NSCLC (13/29, 45%). In contrast, none of 37 small-cell lung cancer (SCLC) cell lines and five extra-pulmonary small-cell cancer cell lines had ras mutations. ras mutations were not correlated with sex of the patients, tumor extent, prior therapy status or in vitro culture time. G to T or A to T transversions were the most common base substitutions, occurring in codons 12 and 61 respectively. We conclude that ras mutations play a role in the pathogenesis of a subset of NSCLC but are not involved in SCLC.

Alleles↗
PubMed1991

A complex pattern of translational initiation and phosphorylation in L-myc proteins.

Molecular analysis of the human proto-oncogene L-myc revealed a complex pattern of gene expression including alternative splicing and polyadenylation site selection of mRNA, giving rise to at least four mRNAs. These mRNAs in turn can code for several proteins. In this report, we characterize and define the origins of the major L-myc proteins. In vitro translation revealed that (i) two L-myc proteins (p59 and p65) were derived through alternative translational initiation at a non-AUG (CUG) site in intron 1 and at an AUG site in exon 2 of L-myc, and that (ii) extensive post-translational phosphorylation of these proteins yielded three additional proteins (p60, p66, and p68). Transfection experiments in rat embryo cells revealed the in vivo existence of this unusual CUG-initiated protein and demonstrated that it possessed transforming activity. Further, immuno-precipitation using high titered anti-L-myc peptide antisera, of two L-myc expressing small-cell lung cancer cell lines revealed three major L-myc proteins (p60, p66 and p68) all of which were derived from extensive phosphorylation of a p59 protein.

Animals↗
PubMed1990

Correlation of in vitro drug-sensitivity testing results with response to chemotherapy and survival in extensive-stage small cell lung cancer: a prospective clinical trial.

We devised a novel clinical protocol for extensive-stage small cell lung cancer (SCLC), selecting chemotherapy whenever possible on the basis of in vitro drug-sensitivity testing (DST) of individual patients' tumor specimens. Most of the specimens were obtained from metastatic sites during routine staging procedures. Increase of tumor cell number by culture in selective media usually was required before DST could be performed. We used the Weisenthal dye exclusion assay to place the seven drugs in rank order and to select the in vitro best regimen (IVBR), a three-drug combination of proved efficacy in SCLC. After initial staging and specimen acquisition, patients received etoposide and cisplatin (primary therapy) and were restaged after 12 weeks. Patients with partial or no responses and those relapsing after a complete response to primary therapy were switched to the IVBR if DST data were available. If DST data were unavailable, an empiric combination, vincristine-doxorubicin-cyclophosphamide, was administered as secondary therapy. Tumor-containing specimens were collected from 60 of the 80 patients (75%). One or more cell lines were established from 28 patients, and DST data were available from 26 patients (33% of total). Several parameters of in vitro drug sensitivity were significantly associated [two-sided P (P2) less than .05] with clinical response to primary therapy and also with response to the IVBR and were marginally associated with length of survival (.07 less than or equal to P2 less than or equal to .08). Sixteen patients (23%) received their IVBR as secondary therapy, and four of these (25%) attained a complete response, compared with three of 43 (7%) who received an empiric regimen (P2 = .16). We concluded that (a) selection of individualized chemotherapy is labor intensive but feasible in extensive-stage SCLC; (b) DST data are associated with clinical response to primary therapy and to secondary therapy with an IVBR; and (c) further observations will be required if we are to determine whether there is a modest therapeutic benefit to administering the IVBR as a secondary therapy.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1990

Frequent inactivation of the retinoblastoma anti-oncogene is restricted to a subset of human tumor cells.

We have used polyclonal anti-synthetic peptide serum to study the role of retinoblastoma gene (RB) inactivation in a variety of human tumor cell lines. Our analysis indicates that inactivation of the RB protein, p105-Rb, is universal in retinoblastoma cells, vindicating the predictions of the Knudson "two-hit" hypothesis. In addition, our analysis has shown that inactivations of the RB gene are nearly as frequent in a more common human tumor, small cell lung carcinoma. One-third of bladder carcinomas surveyed also carry altered or absent p105-Rb. Other human tumors by contrast demonstrate only infrequent inactivation of the RB gene. These results suggest that inactivation of the RB gene is a critical step in the pathogenesis of a subset of human tumors.

Base Sequence↗
PubMed1990

Opioid and nicotine receptors affect growth regulation of human lung cancer cell lines.

Using specific ligands, we find that lung cancer cell lines of diverse histologic types express multiple, high-affinity (Kd = 10(-9)-10(-10) M) membrane receptors for mu, delta, and kappa opioid agonists and for nicotine and alpha-bungarotoxin. These receptors are biologically active because cAMP levels decreased in lung cancer cells after opioid and nicotine application. Nicotine at concentrations (approximately 100 nM) found in the blood of smokers had no effect on in vitro lung cancer cell growth, whereas mu, delta, and kappa opioid agonists at low concentrations (1-100 nM) inhibited lung cancer growth in vitro. We also found that lung cancer cells expressed various combinations of immunoreactive opioid peptides (beta-endorphin, enkephalin, or dynorphin), suggesting the participation of opioids in a negative autocrine loop or tumor-suppressing system. Due to the almost universal exposure of patients with lung cancer to nicotine, we tested whether nicotine affected the response of lung cancer cell growth to opioids and found that nicotine at concentrations of 100-200 nM partially or totally reversed opioid-induced growth inhibition in 9/14 lung cancer cell lines. These in vitro results for lung cancer cells suggest that opioids could function as part of a "tumor suppressor" system and that nicotine can function to circumvent this system in the pathogenesis of lung cancer.

Carcinoma, Non-Small-Cell Lung↗
PubMed1990

Dominant oncogenes and tumor suppressor genes in the pathogenesis of lung cancer.

An understanding of the molecular pathogenesis of lung cancer has evolved from classic cytogenetic studies and the use of restriction fragment length polymorphisms to encompass the definition of specific genetic abnormalities associated with this disease. Activation of the dominant class of oncogenes is frequent, with involvement of the ras and myc families of genes being the best defined. Several examples of inactivation at specific loci exist and have been related to the presence of tumor suppressor genes, most notably the retinoblastoma gene, p53, and a putative gene located on the short arm of chromosome 3. As our understanding of the nature and interactions between these numerous genetic events evolves, new opportunities for early diagnosis, prevention, and treatment will arise.

Animals↗
PubMed1990

A chemical mismatch cleavage method useful for the detection of point mutations in the p53 gene in lung cancer.

Point mutations in genes can be etiologic of pulmonary diseases, as in the case of the inherited disorders alpha-1-antitrypsin deficiency and cystic fibrosis or in the context of dominant and recessive oncogenes in lung cancer. Various methodologies have been developed to screen for single-base mutations. These techniques include direct DNA sequencing, RNase protection, denaturing gradient gel electrophoresis, and chemical mismatch cleavage. The latter method offers the advantages of rapid and efficient analysis of genomic or cDNA and is thus ideally suited to screening applications. Furthermore, all possible single-base changes can theoretically be detected. In the present work, chemical mismatch cleavage was utilized to detect mutations in the p53 gene in small cell and non-small cell lung cancer. This technique was modified by using a two-step, hemi-nested PCR procedure for preparation of target genomic DNAs permitting an expanded target size for analysis. Evaluation by chemical mismatch cleavage of eight p53 cDNAs derived from lung tumors shown to have different mutations by DNA sequencing correctly detected the presence of a point mutation in all instances. Analysis of six additional tumor genomic DNAs with defined mutations in the corresponding p53 cDNAs accurately confirmed the mutation at the level of the genome. The technique also identified codon 72 and intron 6 polymorphisms. Using the intron 6 polymorphism, loss of heterozygosity at the p53 locus in tumor DNA was readily detected by chemical mismatch cleavage. Finally, utilizing this technique for scanning analysis of the p53 gene of uncharacterized lung tumor DNAs, additional mutations were identified in a prospective manner which were confirmed by sequence analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗