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Biomedical subjects

F McCormick

Publications and source records attributed to F McCormick.

At least 181 records · Page 10Linked to original sources
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↗
PubMed1988

Guanosine triphosphatase activating protein (GAP) interacts with the p21 ras effector binding domain.

A cytoplasmic protein that greatly enhances the guanosine triphosphatase (GTPase) activity of N-ras protein but does not affect the activity of oncogenic ras mutants has been recently described. This protein (GAP) is shown here to be ubiquitous in higher eukaryotes and to interact with H-ras as well as with N-ras proteins. To identify the region of ras p21 with which GAP interacts, 21 H-ras mutant proteins were purified and tested for their ability to undergo stimulation of GTPase activity by GAP. Mutations in nonessential regions of H-ras p21 as well as mutations in its carboxyl-terminal domain (residues 165-185) and purine binding region (residues 117 and 119) did not decrease the ability of the protein to respond to GAP. In addition, an antibody against the carboxyl-terminal domain did not block GAP activity, supporting the conclusion that GAP does not interact with this region. Transforming mutations at positions 12, 59, and 61 (the phosphoryl binding region) abolished GTPase stimulation by GAP. Point mutations in the putative effector region of ras p21 (amino acids 35, 36, and 38) were also insensitive to GAP. However, a point mutation at position 39, shown previously not to impair effector function, did not alter GAP-p21 interaction. These results indicate that GAP interaction may be essential for ras p21 biological activity and that it may be a ras effector protein.

Amino Acid Sequence↗
PubMed1988

Functional properties of proteins coded by three human alpha-interferon genes and a pseudogene.

We have characterized the functional properties of four highly purified recombinant human class I alpha-interferon subtypes whose biological activities have not been described previously. We selected biological and biochemical activities that may discriminate between different functions of these molecules. We found that the alpha subtypes could be discriminated only by antiviral-host range specificity and natural killer cell activation. Differences in their antiproliferative activity were cell line dependent. Competitive binding, antiproliferative activity in agar, enhancement of expression of HLA-ABC, elevation of 2'-5'-oligoadenylate synthetase levels and enhancement of phosphorylation of the Mr 69,000 protein kinase did not allow discrimination among the alpha I subtypes on the tested cell lines.

2',5'-Oligoadenylate Synthetase↗
PubMed1988

Analysis of RAS oncogene mutations in human lymphoid malignancies.

We investigated the frequency of mutations activating RAS oncogenes in human lymphoid malignancies, including B- and T-cell-derived acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin lymphoma. By the polymerase chain reaction/oligonucleotide hybridization method, DNA from 178 cases was analyzed for activating mutations involving codons 12 and 61 of the HRAS, KRAS and NRAS genes and codon 13 of the NRAS gene. Mutations involving codons 12 or 13 of the NRAS gene were detected in 6 of 33 cases of acute lymphoblastic leukemia (6/33, 18%), whereas no mutations were found in non-Hodgkin lymphoma or chronic lymphocytic leukemia. Direct nucleotide sequence analysis of polymerase chain reaction products showed that the mutations involved a G----A transition in five of the six cases of acute lymphocytic leukemia. In four cases the mutations seemed to occur in only a fraction of the neoplastic cells, and one case displayed two distinct NRAS mutations, most likely present in two distinct cell populations. These results indicate the following: (i) RAS oncogenes are not found in all types of human malignancies, (ii) significant differences in the frequency of RAS mutations can be found among subtypes of neoplasms derived from the same tissue, (iii) in lymphoid neoplasms the NRAS mutation correlates with the most undifferentiated acute lymphocytic leukemia phenotype, and (iv) NRAS mutations present in only a fraction of malignant cells may result from either the selective loss or the acquisition of mutated alleles during tumor development.

Animals↗
PubMed1988

Analysis of RAS gene mutations in acute myeloid leukemia by polymerase chain reaction and oligonucleotide probes.

In vitro DNA amplification followed by oligonucleotide dot blot analysis were used to study RAS gene mutations in acute myeloid leukemia (AML). Fifty-two presentation AML DNAs were screened for mutations in codons 12, 13, and 61 of NRAS and in codons 12 and 61 of KRAS and HRAS. Fourteen (27%) contained mutations--all in NRAS and predominantly in codon 12. The most common amino acid substitution identified was of glycine by aspartic acid at codon 12 (7/18), with a G----A transition being the most common base change (11/18). No particular correlation was observed between disease subtype and the incidence or type of NRAS mutation. In DNA samples from four patients, 2 NRAS mutations were found to coexist. NIH 3T3 focus-formation assays revealed that in each case the mutations were present in different NRAS alleles. We also report the absence of a mutated RAS gene in relapse DNAs of four patients in which a RAS oncogene had been detected at presentation. These observations suggest that RAS mutations arise as part of the evolution of neoplastic transformation.

DNA, Neoplasm↗
PubMed1988

Localization of phospholipase A2 in normal and ras-transformed cells.

The cellular localization of phospholipase A2 (PLA2) was examined in normal and ras-transformed rat fibroblasts using immunohistochemical techniques. Polyclonal antibodies were generated against porcine pancreatic PLA2 and were affinity purified for use in this study. The antibodies detected a 16-kD band on immunoblots of total cellular proteins from fibroblasts. In cell-free assays of phospholipase A2 activity, the purified antibodies inhibited the bulk of the enzyme activity whereas control IgG preparations had no effect. Immunofluorescence microscopy indicated that PLA2 was diffusely distributed throughout the cell. Increased concentration of PLA2 was detected under membrane ruffles in normal and ras-transformed cells. Specific immunofluorescence staining was also detected on the outer surface of the normal cells. Immunoelectron microscopy demonstrated the increased accumulation of PLA2 in membrane ruffles and also revealed the presence of the enzyme in microvilli and its association with intracellular vesicles. Ultrastructural localization of PLA2 and the ras oncogene protein, using a double immunogold labeling technique, indicated a spatial proximity between PLA2 and ras proteins in the ruffles of ras-transformed cells. The possible role of PLA2 in the structural rearrangements that underlie membrane ruffling is discussed.

Animals↗
PubMed1988

RAS mutations in myelodysplasia detected by amplification, oligonucleotide hybridization, and transformation.

Members of the RAS gene family have been implicated in many neoplasms with activating mutations around amino acid positions 12 and 61. We have assessed the mutational activation of H, K, and NRAS in myelodysplasia (MDS) by polymerase chain reaction and hybridization with synthetic oligonucleotide probes. Using this method, point mutations in codons 12/13 and 61 of these RAS genes were detected in 20 of 50 patients including two with refractory anemia with ringed sideroblasts (RARS). Ten normal individuals had no detectable RAS mutations. In 11 instances, DNA from patients with detectable RAS mutations were shown to register in either NIH3T3 focus-forming or nude mouse tumorigenicity assays. In addition, one patient (RARS) was shown to have an activated NRAS gene detected by a tumorigenicity assay and Southern blot analyses. Two MDS patients had mutations detected in two different RAS genes. DNA from one of these patients was observed to give rise to transformants with activated N and HRAS. Two patients with detectable NRAS mutations in the MDS stage progressed to AML and DNA from the AML stage registered positively in a transformation assay with NRAS activation. These results show that RAS mutations can occur at early, as well as late, stages of leukemic progression. The incidence of RAS mutations appears to be significantly higher in CMML than in the other subgroups (p = 0.02).

Adult↗
PubMed1987

A cytoplasmic protein stimulates normal N-ras p21 GTPase, but does not affect oncogenic mutants.

The role of guanine nucleotides in ras p21 function was determined by using the ability of p21 protein to induce maturation of Xenopus oocytes as a quantitative assay for biological activity. Two oncogenic mutant human N-ras p21 proteins, Asp12 and Val12, actively induced maturation, whereas normal Gly12 p21 was relatively inactive in this assay. Both mutant proteins were found to be associated with guanosine triphosphate (GTP) in vivo. In contrast, Gly12 p21 was predominantly guanosine diphosphate (GDP)-bound because of a dramatic stimulation of Gly12 p21-associated guanosine triphosphatase (GTPase) activity. A cytoplasmic protein was shown to be responsible for this increase in activity. This protein stimulated GTP hydrolysis by purified Gly12 p21 more than 200-fold in vitro, but had no effect on Asp12 or Val12 mutants. A similar factor could be detected in extracts from mammalian cells. It thus appears that, in Xenopus oocytes, this protein maintains normal p21 in a biologically inactive, GDP-bound state through its effect on GTPase activity. Furthermore, it appears that the major effect of position 12 mutations is to prevent this protein from stimulating p21 GTPase activity, thereby allowing these mutants to remain in the active GTP-bound state.

Animals↗
PubMed1987

Ras p21 as a potential mediator of insulin action in Xenopus oocytes.

The oncogene protein product (p21) of the ras gene has been implicated in mediating the effects of a variety of growth factors and hormones. Microinjection of monoclonal antibody 6B7, which is directed against a synthetic peptide corresponding to a highly conserved region of p21 (amino acids 29 to 44) required for p21 function, specifically inhibited Xenopus oocyte maturation induced by incubation with insulin. The inhibition was dose-dependent and specific since (i) the same antibody had no effect on progesterone-induced maturation, (ii) immunoprecipitation and Western blotting indicated that the antibody recognized a single protein of molecular weight 21,000 in oocyte extracts, and (iii) inhibition was not observed with identical concentrations of normal immunoglobulin. Thus, p21 appears to be involved in mediating insulin-induced maturation of Xenopus oocytes. Furthermore, the mechanism may involve phosphorylation of p21, as p21 was found to be a substrate of the insulin receptor kinase.

Animals↗
PubMed1987

Inhibition of cell surface ruffling and fluid-phase pinocytosis by microinjection of anti-ras antibodies into living cells.

Fibroblasts transformed by ras oncogenes display enhanced cell surface ruffling and fluid-phase pinocytotic activities. Microinjection of antibodies that specifically bind the ras proteins into these cells results in the inhibition of these two surface activities. The possible underlying biochemical basis of the influence of the ras proteins on membrane ruffling and pinocytosis and the potential relationship of these two biological activities to membrane signal transduction are discussed.

Animals↗
PubMed1987

Biochemical and biological properties of the human N-ras p21 protein.

We characterized the normal (Gly-12) and two mutant (Asp-12 and Val-12) forms of human N-ras proteins produced by Escherichia coli. No significant differences were found between normal and mutant p21 proteins in their affinities for GTP or GDP. Examination of GTPase activities revealed significant differences between the mutant p21s: the Val-12 mutant retained 12% of wild-type GTPase activity, whereas the Asp-12 mutant retained 43%. Both mutant proteins, however, were equally potent in causing morphological transformation and increased cell motility after their microinjection into quiescent NIH 3T3 cells. This lack of correlation between transforming potency and GTPase activity or guanine nucleotide binding suggests that position 12 mutations affect other aspects of p21 function.

Animals↗
PubMed1986

Origin of short-latency somatosensory evoked potentials to median nerve stimulation in the cat. Comparison of the recording montages and effect of laminectomy.

Short-latency somatosensory evoked potentials were recorded from 23 cats with the frontal-neck, scalp-ear and scalp-noncephalic reference montages. In the frontal-neck recordings, four or five components (n9, n11, n13a, n13b and n14) were identified, whereas three components (p15, p18 and p20) were recorded in the scalp-ear leads. The noncephalic reference recordings had four to six components (p9, p10, p11, p13a, p13b and p14). The origin of these components was investigated by recording direct from the attributed generators and examining the effects of lesions. The suggested generators are as follows: n9, p9 and p10-peripheral nerve; n11, p11-dorsal column; n13a-segmental dorsal horn; p13a-spinocerebellar tract; n13b and p13b-cuneate nucleus and caudal part of the medial lemniscus; n14, p14 and p15-rostral part of the medial lemniscus; p18-thalamocortical radiation; p20-primary somatosensory cortex. Components with similar latencies such as n13a and p13a in the frontal-neck and noncephalic reference recordings had different generators. In the noncephalic reference recordings, the axially orientated dipoles, including the potential produced by the spinocerebellar tract (p13a) were clearly detectable, but the transversely orientated dipole of the segmental dorsal horn (n13a) was indistinct. The frontal-neck montage was distorted by the frontal 'reference' electrode active for part of the axially ascending volleys (p13a in some cats and p14), but could pick up the near-field potentials in the segmental dorsal horn (n13a). Desynchronized volleys in fibre tracts such as the spinothalamic tract did not contribute significantly to the potentials recorded from the skin, whereas the synaptic potential in the cuneate nucleus was shown to have a steep onset and open-field distribution with its dipole orientated in part axially, and was recorded in the noncephalic reference montage. The p9 and p11 positivities fused after laminectomy, suggesting that conductance change at the root entry to the bony spinal canal separates these components in the noncephalic reference recording.

Anesthesia↗