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Biomedical subjects

E W Newcomb

Publications and source records attributed to E W Newcomb.

At least 19 recordsLinked to original sources
PubMed1992

p53 mutations in C57BL/6J murine thymic lymphomas induced by gamma-irradiation and N-methylnitrosourea.

Genomic DNA from thymus tissue obtained from 47 C57BL/6J animals treated with the DNA alkylating agent N-methylnitrosourea or gamma-irradiation were screened for the presence of p53 mutations by using the single strand conformation polymorphism assay. Mutations were detected in 13% (4 of 30) of primary thymic lymphomas but none of 17 early stage lymphomas. The frequency of p53 mutations was the same in tumors induced by N-methylnitrosourea (2 of 15) or by gamma-irradiation (2 of 15). Mutations occurred in the highly conserved regions of the p53 gene in exons 5, 7, and 8. G:C to A:T transitions were commonly observed. One of 4 of the tumors analyzed contained two p53 mutations in exons 7 and 8. A previous study of the same tumors showed that ras mutations occurred with high frequency (greater than 50%) (E. W. Newcomb et al., Cancer Res., 48:5514-5521, 1988). Our data suggest that p53 mutations do not play a major role in carcinogen-induced thymic lymphomas studied here.

Animals↗
PubMed1992

p53 mutations in human malignant gliomas: comparison of loss of heterozygosity with mutation frequency.

Mutations in the p53 gene were analyzed in 40 gliomas using the single strand conformation polymorphism assay together with restriction fragment length polymorphism analysis to assess loss of heterozygosity for 17p alleles in the same tumors. Mutations occurred in 40% of the gliomas and were found in exons 4-8 of the p53 gene. G:C to T:A transversions, which occur in high frequency in some lung (greater than 50%), liver (greater than 80%), breast (30%), and esophageal cancers (25%), were noted in greater than 25% of the gliomas studied here. These transversions were clustered in exon 5 from codons 156 to 168, a region of the p53 gene not previously associated with a high frequency of mutation, and may represent a new hot spot for mutations in certain cancers. The majority of gliomas (27 of 38) analyzed here retained both 17p alleles. The frequency of p53 mutations was 37% in this group of tumors and increased to 64% in tumors with one 17p allele. Allelic loss for chromosome 17p occurred in 4 of 11 gliomas independently of mutations in the p53 gene. Absence of p53 mutations in 36% of the tumors with one 17p allele suggests that a tumor suppressor gene other than p53 may be located on chromosome 17p and involved in progression to malignancy of some gliomas.

Adolescent↗
PubMed1991

p53 mutations in nonastrocytic human brain tumors.

Genomic DNA from 51 primary human brain tumors was screened for the presence of mutations in the tumor suppressor gene, p53, using the polymerase chain reaction and single strand conformation polymorphism analysis, followed by direct DNA sequencing. Mutations leading to an amino acid change were found in 2 of 17 (12%) oligodendrogliomas and 2 of 19 (11%) medulloblastomas but none of 15 ependymomas. Sites of mutations were in exon 5 (codon 141), exon 6 (codon 193 and 213), and exon 7 (codon 246). In addition, there were silent mutations in exon 6 (codon 213) in one oligodendroglioma and in one ependymoma. This study points to the possible role of the p53 tumor suppressor gene in some central nervous system neoplasms of divergent histogenesis.

Adult↗
PubMed1991

Differential expression of the normal and mutated K-ras alleles in chemically induced thymic lymphomas.

The presence of point mutations in the K-ras gene was examined in murine thymic lymphomas induced by a single dose of N-methylnitrosourea by the RNase A mismatch cleavage method and by allelic-specific oligonucleotide hybridization of in vitro amplified DNA by polymerase chain reaction. The results show that the frequency of mutations is lower than that of tumors induced by multiple N-methylnitrosourea treatments. Four mutations identified were the aspartic acid at codon 12, a G:C to A:T transition in its second position. A G:C to T:A transversion in codon 146 was also found in one thymic lymphoma, changing the amino acid alanine to serine. The use of the RNase A assay allowed an estimation of the relative expression levels of both normal and mutant K-ras alleles. The results show that in approximately one half of the tumors the mutant allele is predominantly expressed, suggesting that the normal allele has been lost or that the mutant allele has been amplified relative to the normal. Altogether, these findings are consistent with ras mutations occurring in some instances during tumor development and with a ras effect being not strictly dominant but favoring selection for increasing levels of expression from the oncogenic allele.

Animals↗
PubMed1991

p53 mutations in human lymphoid malignancies: association with Burkitt lymphoma and chronic lymphocytic leukemia.

We have investigated the frequency of p53 mutations in B- and T-cell human lymphoid malignancies, including acute lymphoblastic leukemia, the major subtypes of non-Hodgkin lymphoma, and chronic lymphocytic leukemia. p53 exons 5-9 were studied by using genomic DNA from 197 primary tumors and 27 cell lines by single-strand conformation polymorphism analysis and by direct sequencing of PCR-amplified fragments. Mutations were found associated with (i) Burkitt lymphoma (9/27 biopsies; 17/27 cell lines) and its leukemic counterpart L3-type B-cell acute lymphoblastic leukemia (5/9), both of which also carry activated c-myc oncogenes, and (ii) B-cell chronic lymphocytic leukemia (6/40) and, in particular, its stage of progression known as Richter's transformation (3/7). Mutations were not found at any significant frequency in other types of non-Hodgkin lymphoma or acute lymphoblastic leukemia. In many cases, only the mutated allele was detectable, implying loss of the normal allele. These results suggest that (i) significant differences in the frequency of p53 mutations are present among subtypes of neoplasms derived from the same tissue; (ii) p53 may play a role in tumor progression in B-cell chronic lymphocytic leukemia; (iii) the presence of both p53 loss/inactivation and c-myc oncogene activation may be important in the pathogenesis of Burkitt lymphoma and its leukemic form L3-type B-cell acute lymphoblastic leukemia.

Base Sequence↗
PubMed1991

ras activation in human tumors and in animal model systems.

Environmental agents such as radiation and chemicals are known to cause genetic damage. Alterations in a limited set of cellular genes called proto-oncogenes lead to unregulated proliferation and differentiation. We have studied the role of the ras gene family in carcinogenesis using two different animal models. In one case, thymic lymphomas were induced in mice by either gamma or neutron radiation, and in the other, keratoacanthomas were induced in rabbit skin with dimethylbezanthracene. Human keratoacanthomas similar to the ones induced in rabbits were also analyzed. We found that different types of radiation such as gamma rays and neutrons, induced different point mutations in ras genes. A novel K-ras mutation in codon 146 has been found in thymic lymphomas induced by neutrons. Keratoacanthomas induced in rabbit skin by dimethylbenzanthracene show a high frequency of H-ras-activated genes carrying a mutation in codon 61. The same is observed in human keratoacanthomas, although mutations are in both the 12th and the 61st codons of the H-ras gene. H-ras activation is less frequent in human squamous cell carcinomas than in keratoacanthomas, suggesting that ras genes could play a role in vivo in differentiation as well as in proliferation.

3T3 Cells↗
PubMed1990

Neutron radiation can activate K-ras via a point mutation in codon 146 and induces a different spectrum of ras mutations than does gamma radiation.

Neutron radiation is known to produce tumors in animals and cause cell transformation. We have developed a protocol to efficiently induce thymic lymphomas in RF/J mice by a single acute dose of neutron irradiation. Activated ras genes were detected in 17% (4 of 24) of the tumors analyzed. One of the tumors contained a K-ras gene activated by a point mutation in codon 146. Activating ras mutations at position 146 have not been previously detected in any known human or animal tumors. The spectrum of ras mutations detected in neutron radiation-induced thymic lymphomas was different from that seen in thymic lymphomas induced by gamma radiation in the same strain of mice. These results may have important implications for the mechanisms by which different types of radiation damage DNA.

Animals↗
PubMed1990

Comparative analysis and anatomic distribution of ras p21, IL-2R, and MEL-14 in malignant and hyperplastic murine thymus.

The distribution and localization of thymocytes positive for p21 ras, the lymphocyte homing receptor antigen MEL-14, and IL-2 receptors were studied by immunohistology and flow cytometry. Comparisons were made between age-matched normal mice, carcinogen-treated mice at early (stage II) and late (stage III) stages of disease, and cortisone-treated mice. In normal thymus, the majority of cortical and medullary thymocytes are p21 ras positive. MEL-14hi- and IL-2R-positive cells are located in the cortex and comprise less than 5% of the thymus population. Stage II carcinogen-treated animals consistently show increased numbers of MEL-14hi cells in the thymus, with fewer animals having increased numbers of IL-2R positive cells. These populations appear to be different from one another. All stage III animals have MEL-14hi-positive tumor cells, which in 70% of the cases also express IL-2R. Cortisone treatment was used to study non-malignant proliferation. After cortisone treatment there is a marked increase of p21 ras staining in both the cortex and medulla during the first 72-hour interval. Within 24 hours, 50% of the thymocytes are IL-2R positive, but MEL-14hi cells are not detected. By 48 hours, 90% of the thymus population expresses IL-2R and 50% of the cells are MEL-14hi positive, and this results in a substantial population of cells positive for both IL-2R positive:MEL-14hi markers. This population rapidly disappears by 72 hours, leaving 90% of the cells MEL-14hi positive and less than 10% IL-2R positive. The staining of p21 ras at 72 hours is unusual, showing a speckled, cytoplasmic pattern. In light of our findings, we propose that the first step in thymic lymphomagenesis in carcinogen-treated C57BL/6 mice involves the rare cortical MEL-14hi subpopulation and is thymic dependent. A late stage involves expression of IL-2 receptors by a subset of MEL-14hi cells, thus conferring the potential for autonomous growth and malignancy.

Animals↗
PubMed1989

Transformation and plasmacytoid differentiation of EBV-infected human B lymphoblasts by ras oncogenes.

The biological effects of ras oncogene activation in B cells were studied by using amphotropic retroviral vectors to introduce H- or N-ras oncogenes into human B lymphoblasts immortalized by Epstein-Barr virus. Expression of both H- and N-ras oncogenes led to malignant transformation of these cells, as shown by clonogenicity in semisolid media and tumorigenicity in immunodeficient mice. In addition, terminal differentiation into plasma cells was detectable as specific changes in morphology, immunoglobulin secretion, and cell surface antigen expression. This combined effect, promoting growth and differentiation in human lymphoblasts, represents a novel biological action of ras oncogenes and has implications for the pathogenesis of terminally differentiated B-lymphoid malignancies such as multiple myeloma.

Animals↗
PubMed1989

Radiation and chemical activation of ras oncogenes in different mouse strains.

A survey of a large series of radiation- or chemically induced thymic lymphomas in (AKR X RF)F1, RF/J, 129/J, and C57BL/6J mouse strains for activated ras oncogenes showed that of the tumors containing transforming activity, in more than 75% of the cases this activity segregated with either K-ras or the N-ras gene. H-ras activity was never detected. The genetic background of the host influenced susceptibility to tumor induction and oncogene activation. The K-ras gene was preferentially activated over the N-ras gene (approximately 2:1) whether the inducing agent was radiation or the chemical N-nitrosomethylurea. The activating mutation for the K-ras gene was consistently identified as a GGT to GAT transition in codon 12. In contrast, several different mutations of the N-ras gene were identified and localized to codons 12, 13, or 61. Assessment of the allelic composition of the ras locus shows that some proportion of the tumors lost the normal ras allele.

Animals↗
PubMed1988

ras oncogenes and phenotypic staging in N-methylnitrosourea- and gamma-irradiation-induced thymic lymphomas in C57BL/6J mice.

We have investigated stages of thymic lymphoma development in radiation and N-methylnitrosourea (NMU)-treated C57BL/6J mice. The lymphoma cell was identified serologically as a cortical population bearing MEL-14hi, H-2Khi, and IL-2R+ surface markers. According to these parameters in C57BL/6J mice the lymphoma cell was the same regardless of inducing agent or activated oncogene (ras or non-ras). Transforming activity in the radiation and NMU-induced tumors was analyzed using both the nude mouse tumorigenicity assay and the focus-forming assay. 8/10 NMU-induced tumors and 12/15 radiation-induced tumors showed transforming activity in the tumorigenicity assay. Southern blot analysis of the nude mouse transformants demonstrated K-ras transforming sequences in eight of eight NMU-induced lymphoma DNAs, two of 12 radiation-induced lymphoma DNAs and N-ras transforming sequences in five of 12 radiation-induced lymphoma DNAs. The non-ras transforming activity in five DNAs from radiation-induced thymic lymphomas indicates the presence of an unidentified oncogene(s) in these tumors. Staging of thymic lymphoma development in this animal model system will allow the study of oncogene activation early in the course of carcinogen-induced disease. These results also emphasize the high sensitivity of the nude mouse assay to score for activated oncogenes and might also indicate a high frequency of K-ras activation in NMU-induced lymphomas in C57BL/6J mice.

Animals↗
PubMed1988

Identification of a specific marker chromosome early in tumor development in gamma-irradiated C57BL/6J mice.

Chromosomal changes such as aneuploidies, translocations, and gene amplification occur in many murine tumors. In this study, we have analyzed the changes in chromosomes at different stages of tumor development in C57BL/6J mice treated with gamma-irradiation or the chemical carcinogen, N-methylnitrosourea. Trisomy 15 occurred in both groups of mice regardless of inducing agent. The frequency of this event differed significantly in radiation-treated animals between stage I and stage II of the disease. A specific marker chromosome occurred only in the gamma-irradiated mice and not in the mice treated with N-methylnitrosourea. This marker consists of a translocation between chromosomes 1 and 5. It occurred in 43% of the gamma-irradiated animals at stage 1 of the disease and did not vary markedly during tumor development. In contrast, trisomy 15 increased in frequency between stages I and II of the disease in the same animals. These results suggest that the translocation event may be an early event in tumor development, whereas trisomy 15 may contribute to tumor progression.

Animals↗
PubMed1987

Pathogenesis of Burkitt lymphoma: expression of an activated c-myc oncogene causes the tumorigenic conversion of EBV-infected human B lymphoblasts.

To study the pathogenesis of Burkitt lymphoma, we introduced activated c-myc genes into human EBV-infected lymphoblastoid cells derived from in vitro infection of normal cord blood or directly from infected peripheral blood from AIDS patients. In both cell types the constitutive expression of exogenous c-myc caused negative regulation of endogenous c-myc expression, changes in growth properties typical of transformed cells, and acquisition of tumorigenicity in immunodeficient mice. In all myc-transfected populations the degree of malignancy directly correlated with the level of c-myc mRNA. EBV infection and c-myc activation are thus sufficient for the tumorigenic conversion of human B cells in vitro, strongly supporting the hypothesis that these same two pathogenetic steps may be involved in the in vivo development of Burkitt lymphoma.

B-Lymphocytes↗
PubMed1986

Oncogene activation and surface markers in mouse lymphomas induced by radiation and nitrosomethylurea.

Thymic lymphomas have been induced by gamma-radiation and treatment with the chemical nitrosomethylurea in different mice strains. As indicated by the NIH 3T3 focus forming assay, a significant percentage of the tumors contain activated oncogenes of the ras family (K or N). Cloning and sequencing has enabled us to identify single base mutations as the only significant alteration present in the activated oncogenes. These alterations result in the substitution of amino-acid 12 or 61 of the p21 product of the ras genes. With the use of synthetic oligonucleotides it has been found that the tumors do not all contain the same mutation and in one case so far the normal allele is absent.

Animals↗
PubMed1985

A comparative analysis of radiation- and virus-induced leukemias in BALB/c mice.

Endogenous murine leukemia virus (MuLV) proviral copies were analyzed in thymomas induced in normal BALB/c (Fv-1b) and in Fv-1n congenic mice by X-irradiation. Both strains of mice developed leukemia with similar kinetics, indicating that N-tropism of endogenous MuLV was not a rate-limiting factor in development of disease. Southern blot analysis, using a probe specific for ecotropic virus and for ecotropic-specific sequences retained in pathogenic, env-recombinant viruses, showed that the majority of radiation leukemias lacked newly acquired, clonally integrated, proviruses. This was in contrast to virus-induced leukemias, which routinely exhibited several new proviral integration sites. When an internal proviral DNA restriction fragment was monitored, some radiation leukemias showed evidence of nonclonal infection, accounting for more frequent isolation of infectious virus from such leukemias. Differences in expression of T-cell surface antigens were found in X-ray-induced and virus-induced leukemias. All radiation leukemias were TL positive, whereas virus-induced leukemias were primarily negative for TL. Some differences were also found in Lyt-1 and Lyt-2 expression. The data as a whole suggest that, in the majority of cases, radiation leukemogenesis is not initiated by a viral route--that is, the sort of viral mechanism for which exogenous infection by known pathogenic MuLV is the paradigm.

Animals↗