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Biomedical subjects

F McCormick

Publications and source records attributed to F McCormick.

At least 163 records · Page 9Linked to original sources
PubMed1990

Biochemical characterization of baculovirus-expressed rap1A/Krev-1 and its regulation by GTPase-activating proteins.

Normal human rap1A and 35A rap1A (which encodes a protein with a Thr-35----Ala mutation) were cloned into a baculovirus transfer vector and expressed in Sf9 insect cells. The resulting proteins were purified, and their nucleotide binding, GTPase activities, and responsiveness to GTPase-activating proteins (GAPs) were characterized and compared with those of Rap1 purified from human neutrophils. Recombinant wild-type Rap1A bound GTP gamma S, GTP, and GDP with affinities similar to those observed for neutrophil Rap1 protein. The rate of exchange of GTP by Rap1 without Mg2+ was much slower than that by Ras. The basal GTPase activities by both recombinant proteins were lower than that observed with the neutrophil Rap1, but the GTPase activity of the neutrophil and wild-type recombinant Rap1 proteins could be stimulated to similar levels by Rap-GAP activity in neutrophil cytosol. In contrast to wild-type Rap1A, the GTPase activity of 35A Rap was unresponsive to Rap-GAP stimulation. Neither recombinant Rap1A nor neutrophil Rap1 protein GTPase activity could be stimulated by recombinant Ras-GAP at a concentration 25-fold higher than that required to hydrolyze 50% of H-Ras-bound GTP under similar conditions. These results suggest that the putative effector domains (amino acids 32 to 40) shared between Rap1 and Ras are functionally similar and interact with their respective GAPs. However, although Rap1 and Ras are identical in this region, secondary structure or additional regions must confer the ability to respond to GAPs.

Animals↗
PubMed1990

Clinical characteristics of acromegalic patients whose pituitary tumors contain mutant Gs protein.

Activating mutations in the gene for the alpha-chain of Gs, the stimulatory regulator of adenylyl cyclase, have been identified in human GH-secreting pituitary tumors. Using the polymerase chain reaction and allele-specific oligonucleotide hybridization, we screened 25 GH-secreting tumors for the presence of the activating mutations. We also reviewed the clinical charts of the patients from whom the tumors were removed. Of 25 tumors, 10 (40%) contained activating mutations. Patients in the mutation-positive group came to surgery with smaller tumors and had lower GH levels. The activating mutations identify a subgroup of GH-secreting pituitary tumors that probably arise from a shared oncogenic mechanism.

Acromegaly↗
PubMed1990

RAS mutations in patients following cytotoxic therapy for lymphoma.

The occurrence of Myelodysplastic Syndrome (MDS) and Acute Myeloblastic Leukaemia (AML) following cytotoxic therapy for neoplastic disease is well recognised. RAS mutations are common in patients with MDS and AML. To determine whether these lesions are found as early markers of secondary disease, we have studied the incidence of RAS mutations in the peripheral blood of 70 patients in complete remission from lymphoma. Patients were treated by standard chemotherapy regimes and/or localised radiotherapy. Treatment had been given 6 months to 14 1/2 years previously and no patient showed any sign of residual disease. Genomic DNA from peripheral blood leukocytes was amplified in vitro at target codons of N, K and H RAS genes, and mutations detected by hybridisation with oligonucleotide probes. RAS mutations were detected in 9 subjects. One patient with an N12 valine (Val) substitution had been in complete remission from Hodgkin's disease (HD) for 9 years. DNA from this patient registered in a nude mouse tumorigenicity assay (NMT). The N12 Val mutation was not detected in the original tumour tissue from the same patient. A second patient in remission from HD showed evidence of co-existent N12 cysteine (Cys) and N13 valine (Val) substitutions which were not detected in presentation material or unaffected tissues. All patients are currently haematologically normal, indicating that clones of mutant RAS bearing cells may be detected prior to any overt sign of disease.

Animals↗
PubMed1990

Retrospective analysis of ras gene activation in myeloid leukemic cells.

Ras genes are activated by point mutations at critical sites of their coding regions. Activated N-ras genes with transforming ability have been detected in patients with myelodysplastic syndromes (MDS), acute myelogenous leukemia (AML) and in human myeloid cell lines. We used polymerase chain reaction (PCR), differential oligonucleotide hybridization and direct DNA sequencing to retrospectively analyze the N-ras gene of blast cells from the same patient (a) at time of diagnosis of MDS, (b) after the patient had developed AML. Two types of archival tissue samples served as a source of cells. Different passages of the KG-1 myeloid cell line which had been established from leukemic blasts of this patient were also analyzed. We found that native blast cells isolated at either of the two disease stages did not carry an N-ras mutation, and neither did early passage KG-1 cells. However, direct DNA sequencing of PCR-amplified DNA from nude mice transformants induced by DNA from late passage of the KG-1 cell line revealed two linked mutations involving both the second nucleotide of codon 12 and the third nucleotide of codon 15 of N-ras. The nucleotide substitution at codon 15 did not result in an amino acid substitution (silent mutation). The mutations probably occurred during prolonged passaging of the KG-1 cells and might have been overlooked by oligonucleotide hybridization assay.

Animals↗
PubMed1990

Oncogenes in multiple myeloma: point mutation of N-ras.

Alterations of ras, c-myc and bcl-1 have been described in hematologic malignancies of lymphoid origin. We investigated the structure of these genes and evaluated the frequency of point mutations involving H-, K- or N-ras in bone marrow samples from patients with multiple myeloma. No abnormalities were detected in the c-myc and bcl-1 genes, but two of 17 patients were found to have N-ras mutations by differential oligonucleotide hybridization and dideoxynucleotide sequencing following amplification by polymerase chain reaction. Bone marrow DNA from both patients had identical missense mutations of N-ras codon 61 changing CAA to AAA, resulting in a substitution of lysine for glutamine in the encoded protein. Multiple myeloma is the first mature B cell neoplasm found to harbor ras mutations.

Base Sequence↗
PubMed1989

Genetic analysis of mammalian GAP expressed in yeast.

We have designed a vector to express the mammalian GAP protein in the yeast S. cerevisiae. When expressed in yeast, GAP inhibits the function of the human H-rasgly12 protein, but not that of the H-rasval12 protein, and complements the loss of IRA1. IRA1 is a yeast gene that encodes a protein with homology to GAP and acts upstream of RAS. Mammalian GAP can therefore function in yeast and interact with yeast RAS. Because expression of GAP complements ira1-mutants, we propose that GAP shares some biochemical functions with IRA1. Other studies indicate that IRA1 controls the level of RAS activity, presumably by regulating GTP hydrolysis. By analogy, we propose that GAP may play a similar role.

Cloning, Molecular↗
PubMed1989

Production of and responsiveness to transforming growth factor-beta in normal and oncogene-transformed human mammary epithelial cells.

Transforming growth factors-beta (TGF beta) are a family of closely related, ubiquitously expressed growth factors with the common properties of induction of growth inhibition and expression of differentiation-related markers in epithelial cells. We investigated the role of TGF beta 1 in growth regulation of normal human mammary epithelial cells and in benzo(a)pyrene immortalized sublines further transformed by oncogenes in retroviral vectors. The normal cells were markedly growth inhibited by TGF beta 1, produced TGF beta in a latent form, and expressed TGF beta receptors. In the immortalized cells, both TGF beta-induced growth inhibition and TGF beta receptor binding were reduced. With the single oncogenes v-Ha-ras, v-mos, and SV40 T, growth sensitivity to TGF beta 1 increased, but TGF beta production or TGF beta receptor expression was not altered. Transformation to full malignancy by both SV40 T and v-Ha-ras led to escape from growth inhibition by TGF beta under anchorage-independent, but not anchorage-dependent, conditions without affecting TGF beta production or receptor characteristics. Thus, modulation of TGF beta growth responsiveness in these normal and oncogene transformed human mammary epithelial cells apparently occurs at a level distal to TGF beta receptor binding and is not solely correlated to expression of transforming oncogenes. Further, modulation of TGF beta production is not an indicator of malignant transformation in this system.

Benzo(a)pyrene↗
PubMed1989

Vimentin rather than keratin expression in some hormone-independent breast cancer cell lines and in oncogene-transformed mammary epithelial cells.

To characterize differences in gene expression between hormone-dependent and hormone-independent mammary carcinoma, we cloned complementary DNAs of genes expressed in a hormone-independent breast carcinoma cell line that were not expressed in a hormone-dependent line. One clone, which was isolated in many copies, coded for the intermediate filament protein vimentin. A complementary DNA clone 1.8 kilobases long included the entire protein-coding region for vimentin. Vimentin was expressed by more than one-half of the hormone-independent breast carcinoma cell lines tested but not by the hormone-dependent cell lines. The cell lines which expressed vimentin expressed only low levels of cytokeratins. The correlation between vimentin expression and more advanced stages of mammary cell transformation was tested in a model system in which immortal, nontumorigenic human mammary epithelial cells or derivative lines transformed with v-ras-H or SV40 T-antigen were found not to express vimentin, whereas a derivative highly tumorigenic cell line transformed by both v-ras-H and T-antigen did express vimentin. Analysis of several other kinds of epithelial carcinoma cell lines showed only rare examples of vimentin expression.

Base Sequence↗
PubMed1989

Human bladder and colon carcinomas contain activated ras p21. Specific detection of twelfth codon mutants.

Members of the ras family of proto-oncogenes code for 21,000-dalton molecular weight protein products (p21s). Transformation of cells from the normal to the malignant phenotype in experimental studies has been associated with point mutations within the coding region for these ras proteins. Recent reports demonstrate that 40% of human colon cancers and 20% of acute leukemias contain ras mutations in the twelfth or thirteenth codon that can result in amino acid substitutions at these positions in the p21 products. Similarly, studies of ras mRNA detected 40% of human colon tumors with twelfth codon c-Ki-ras mutant mRNA. The authors previously developed a nonradioactive double-antibody enzyme-linked immunoblot assay (ELIBA) for detection of normal and mutant ras p21. They have adapted that technology to specifically detect twelfth codon activated ras p21 utilizing mutation-specific antisera. In this report the authors show that one of seven de novo human bladder cancers and four of seven colon cancers express a twelfth codon activated ras p21. These results document that mutations at both the DNA and mRNA levels are ultimately translated into an abnormal protein product present in human tumors.

Blotting, Western↗
PubMed1989

ras GTPase activating protein: signal transmitter and signal terminator.

Evidence that GAP is an effector of ras action can be summarized as follows: GAP interacts at a site on p21 defined genetically as the effector binding site. Regions of p21 that are nonessential for biological activity are nonessential for GAP interaction. GAP interacts with all known types of p21. (Upstream factors are expected to be specific for individual types). GAP interacts with p21 proteins (normal and mutant) in a GTP-dependent fashion. None of these constitute proof. It remains possible that GAP simply regulates p21-GTP levels, and binds to the same site as the true effector without transmitting a downstream signal. If indeed GAP mediates ras action, the question immediately arises as to the biochemical function of GAP itself. The requirement of ras proteins for membrane localization to exert their effects may be a valuable clue in the search for this function. Perhaps GAP is an enzyme (or is bound to an enzyme) that acts on membrane components in a p21-GTP-dependent manner and in doing so transmits signals to other downstream effectors. The ability of GAP to interact with many members of the ras family would allow many upstream signals to feed into this downstream pathway. Clearly, proof (or disproof) that GAP is downstream of ras is the next step toward clarification of this aspect of ras action; identification of biochemical activities associated with GAP (or the true ras effector) will, we hope, follow soon.

Animals↗
PubMed1989

Identification of resonances from an oncogenic activating locus of human N-RAS-encoded p21 protein using isotope-edited NMR.

A sample of Escherichia coli-expressed human N-RAS-encoded p21, a 21-kDa protein, was selectively labeled with 15N at each of the 14 glycine amide positions. Two-dimensional proton-observe 15N correlation spectra showed one peak for each glycine residue. Five glycine resonances were identified with residues near the nucleotide binding site and provide useful reporters of several oncogene-activating positions. Three of these resonances were assigned to residues 10, 15, and 115 from the spectrum of a sample that was also labeled with [13C]valine. These resonances showed extra splitting or broadening due to the 13C label, which could be eliminated by 13C decoupling. Two other peaks were unambiguously identified as Gly-12 and Gly-13 using a one-dimensional edited nuclear Overhauser experiment and by spectral comparison with an Asp-12 mutant. These assignments have provided several site-specific probes of critical domains in p21.

Carbon Isotopes↗
PubMed1989

Transforming growth factor alpha production and epidermal growth factor receptor expression in normal and oncogene transformed human mammary epithelial cells.

We have characterized the expression of transforming growth factor alpha (TGF alpha) and its receptor, the epidermal growth factor receptor (EGF-R), in normal and malignantly transformed human mammary epithelial cells. Human mammary epithelial cells were derived from a reduction mammoplasty (184), immortalized by benzo-a-pyrene (184A 1N4), and further transformed by the oncogenes simian virus 40 T (SV40 T), v-Ha-ras, and v-mos alone or in combination using retroviral vectors. 184 and 184A 1N4 cells require EGF for anchorage-dependent clonal growth. In mass culture, they secrete TGF alpha at high concentrations and exhibit an attenuated requirement for exogenous EGF/TGF alpha. SV40 T transformed cells have 4-fold increased EGF-R, have acquired the ability to clone in soft agar with EGF/TGF alpha supplementation, but are not tumorigenic. Cells transformed by v-mos or v-Ha-ras are weakly tumorigenic and capable of both anchorage dependent and independent growth in the absence of EGF/TGF alpha. Cells transformed by both SV40 T and v-Ha-ras are highly tumorigenic, are refractory to EGF/TGF alpha, and clone with high efficiency in soft agar. The expression of v-Ha-ras is associated with a loss of the high (but not low) affinity binding component of the EGF-R. Malignant transformation and loss of TGF alpha/EGF responsiveness did not correlate with an increase in TGF alpha production. Thus, TGF alpha production does not appear to be a tumor specific marker for human mammary epithelial cells. Differential growth responses to EGF/TGF alpha, rather than enhanced production of TGF alpha, may determine the transition from normal to malignant human breast epithelium.

Animals↗
PubMed1989

The polymerase chain reaction and cancer diagnosis.

The introduction of polymerase chain reaction (PCR) technology, and its continual refinement, have had an enormous impact on many areas of molecular biology, including cancer research. This review examines several types of PCR assays that have been designed to investigate the molecular basis of cancer, in particular, those assays that have potential diagnostic applications.

Gene Amplification↗
PubMed1989

ras mutations in human melanotic lesions: K-ras activation is a frequent and early event in melanoma development.

We have used polymerase chain reaction (PCR), an amplification procedure, and oligonucleotide hybridization to detect ras gene point mutations in DNA from melanoma tumor samples. Genomic DNA was examined from 40 specimens of melanotic lesions, including benign nevi, primary melanomas, lymph node metastases, and systemic metastases. Adjacent normal skin or peripheral blood was analyzed as control material in 28 cases. ras mutations were detected overall in 25% of malignant tumors. In addition, mutations of all three ras genes were detected. We observed ras mutations in 2 of 4 benign atypical nevi (2 X K12), 4 of 22 primary melanomas (3 X K12, 1 X H12, 1 X N61), and 4 of 14 secondary (5 X K12, 1 X N61) tumors. One with a primary melanoma had concurrent K12 and H12, and two patients with secondary tumors had concurrent K12/N61 and K12 Asp/K12 Val mutations, respectively, making a total of 10 of 40 (25%) patients with ras mutations. This is the first demonstration of K-ras mutations in human melanoma. The presence of K-ras mutations in nevi, putative melanoma precursors, suggests that ras activation may be an early event in melanoma development. No correlation between tumor thickness and the presence of a ras mutation was observed.

Adult↗
PubMed1988

Transformation of human mammary epithelial cells by oncogenic retroviruses.

We have introduced viral oncogenes into human mammary epithelial cells through the use of murine retroviruses. A continuous cell line (184A1N4) derived from benzo(a)pyrene treatment of normal breast epithelial cells was used as a recipient for the ras, mos, and T-antigen oncogenes. Each of these oncogenes enabled the 184A1N4 cells to grow in a selective medium, thus demonstrating the potential utility of these cells for oncogene detection and isolation. 184A1N4 cells transformed by T-antigen were nontumorigenic in athymic mice, but v-ras transformants were weakly tumorigenic. Transformants bearing both the T-antigen and ras oncogenes were strongly tumorigenic, however. The karyotype of these double transformants shows a high degree of stability. These results demonstrate the stepwise acquisition of the fully malignant phenotype by normal human epithelial cells in vitro.

Animals↗