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Biomedical subjects

P B Rowe

Publications and source records attributed to P B Rowe.

At least 19 recordsLinked to original sources
PubMed1995

Isolation of a novel macrophage-specific gene by differential cDNA analysis.

To analyze myelomonocytic differentiation we have used the approach of differential cDNA analysis to isolate novel genes that are preferentially expressed in mature macrophages. Differential screening of a macrophage cDNA library led to the identification of a novel cDNA that showed macrophage lineage- and differentiation stage-specific expression. Transcripts from the gene, which we have termed Mpg-1, are found at a high level in mature human and murine macrophages and at a moderate level in certain myelomonocytic cell lines. The expression of Mpg-1 was found to increase when murine fetal liver hematopoietic progenitor cells were induced to differentiate into macrophages. An Mpg-1-specific transcript was not detected in a wide variety of other tissues and cell lines. The DNA sequence of Mpg-1 (4,214 bp) was obtained from a series of overlapping cDNA, 3' rapid amplification of cDNA ends (RACE), and genomic clones. Primer extension analysis predicted the existence of multiple transcription start sites, ranging from 26 to 117 bp upstream of the 5' proximal ATG of the open reading frame. The predicted 669-amino acid, Mpg-1-encoded protein has potential glycosylation and phosphorylation sites in addition to a signal sequence. The core protein is predicted to have a molecular weight of 71 to 74 kD. Computer-assisted local similarity searches indicate that Mpg-1 is a novel gene that may share a distant ancestry to perforin, a lytic protein found in cytotoxic T lymphocytes and natural killer cells.

Amino Acid Sequence↗
PubMed1994

In vitro transformation of Li-Fraumeni syndrome fibroblasts by SV40 large T antigen mutants.

Transfection of SV40 early region DNA into normal human diploid fibroblasts (NHDFs) increases their proliferative potential to a limited extent. We have investigated the roles of the SV40 large T antigen (LTAg) regions responsible for binding to the protein products of the retinoblastoma (Rb) and p53 genes in this temporary escape from senescence. Patients encoding LTAg mutants were transfected into NHDFs and into Li-Fraumeni syndrome (LFS) fibroblasts which are heterozygous wild-type (wt)/null-mutant for p53. A LTAg mutated in the p53-binding region (T402DE) had greatly reduced efficiency of focus formation, and a p110Rb-binding mutant was unable to induce any foci. T402DE-induced NHDF foci senesced at the same time as untransfected cells, but the equivalent LFS foci all had increased proliferative potentials, with the greatest increase being seen in clones that lost the wt p53 allele. One LFS clone expressed the T402DE mutant during focus formation, but later lost both the T402DE DNA and the wt p53 allele. We conclude that SV40-induced focus formation in NHDFs requires the LTAg p110Rb-binding region, and is enhanced by loss of normal p53 function. In contrast, increased proliferative potential is primarily due to loss of p53 function.

Antigens, Polyomavirus Transforming↗
PubMed1993

Flow cytometric DNA analyses of frozen samples from children's solid tumors.

Cell ploidy and proliferative activity may be useful to clinicians in treating children with solid tumors. Frozen specimens from children with malignant solid tumors were tested at the time of diagnosis for ploidy and proliferative activity. The flow cytometric DNA histograms were examined to assess the sensitivity of the tumor cell preparation and staining method. All the frozen tumors received (n = 58), from children aged 1 mth to 17 yrs, were analyzed and included in this study. The more common tumors were neuroblastomas (n = 21) and Wilms' tumors (n = 16). The majority of tumors (91%) exhibited a diploid peak from the significant proportion of stromal/epithelial cells in the tumor specimen. The tightness of the diploid G0G1 peaks as measured by their coefficient of variations (CV) produced a mean CV of 2.43% +/- SD 0.62, range 1.3% to 4.6%. The G0G1 CVs from the aneuploid peaks had a mean of 2.34% +/- SD 0.68 with the range 0.5% to 3.7%. There was, however, a proportion of tumors (9%) which showed no normal "diploid" peak on the histogram. These tumor cell suspensions had insignificant numbers of diploid cells. Unless the position of the diploid peak could be identified on the DNA histogram the ploidy result from such tumors would be incorrect. Reference diploid cells can be added to cell suspensions prepared from frozen tumors in order to identify the position of the G0G1 peak. Reference diploid cells cannot be added to single cell preparations from paraffin embedded specimens, therefore making it impossible to define the diploid peak position in these tumors.(ABSTRACT TRUNCATED AT 250 WORDS)

Child↗
PubMed1992

Both myeloproliferative disease and leukemia are induced by transplantation of bone marrow cells expressing v-myc.

An in vivo system has been established to investigate v-myc-induced hematopoietic neoplasia in mice. A Moloney murine leukemia virus (Mo-MLV)-derived recombinant retrovirus containing v-myc was used to infect immature bone marrow cells, and these cells were then transplanted into lethally irradiated recipients. All provirus-positive reconstituted mice were found to develop hematopoietic proliferative disorders and, in certain cases, overt leukemia--myeloblastic, myelomonocytic and T lymphocytic. In all cases expression of v-myc was high and the disease type did not correlate with the level of expression. We have isolated immortalized monocytes, myeloid progenitors and T lymphocytes from several of these mice and shown tumorigenicity in secondary syngeneic recipients. This system provides a model for investigating the progression from a pre-leukemic disease to malignancy. In addition, we describe a recombinant v-myc-containing retrovirus that directs high-level v-myc expression from the Mo-MLV promoter in all the hematopoietic cell types examined.

Animals↗
PubMed1992

Germ-line splicing mutation of the p53 gene in a cancer-prone family.

Li-Fraumeni syndrome is a rare autosomal dominant susceptibility to a variety of cancers including carcinomas of the breast and the adrenal cortex, tumors of brain and muscle tissue, and leukemias. Affected individuals develop cancer at a young age and often at multiple primary sites. A study has been conducted into the genetic basis of cancer in a particular Li-Fraumeni syndrome family. Examination of p53 as a candidate susceptibility gene revealed that, in two affected individuals, there was an aberrant larger transcript of 3.6 kilobases present in both tumor and constitutional material in addition to the normal-sized 2.8-kilobase transcript. The additional transcript was not found in three unaffected family members. S1 nuclease mapping localized the insertion toward the 5' end of the p53 transcript near exons 4 and 5, and sequencing revealed a point mutation in the splice donor site of intron 4 in the germ-line of the two affected individuals, which accounted for the presence of the larger transcript. The same splicing mutation was also detected in two obligate carriers and was not found in two unaffected individuals. As no mutations were detected in exons 5-8 in either tumor examined, the second p53 allele was most likely lost during tumorigenesis in both tumors. The demonstration of a germ-line splicing mutation in affected individuals from a Li-Fraumeni syndrome family provides for a novel mechanism of p53 inactivation not seen previously in other affected families, in whom the mutations have all been missense.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Cortex Neoplasms↗
PubMed1991

Establishment in culture and characterization of a strain with mast cell and monocytic properties from the bone marrow of a child with diffuse cutaneous mastocytosis.

Bone marrow was isolated from a child with congenital mastocytosis. Upon prolonged in vitro culture, initially in the presence of interleukin-3 (IL-3), a population of relatively large fusiform, strongly adherent cells grew out plus a subpopulation of smaller nonadherent cells. The morphology of the adherent cells was not typical of fibroblasts, epithelial cells, nor of standard hematopoietic cell types, whereas the morphology of the nonadherent cells resembled mast cells. Neither cell type required the presence of IL-3 nor a feeder layer of fibroblasts for continued growth. Attempts to isolate the two populations were unsuccessful. This cell strain comprised of both cell populations has been termed human bone marrow-derived mastocytosis cells (HBM-M). These cells were found to possess some of the cytochemical, ultrastructural, and surface phenotypic features of degranulated mast cells. They reacted with the mast cell marker, monoclonal antibody YB5.B8, but not with the basophil specific monoclonal antibody Bsp-1 and released the inflammatory mediators histamine, leukotriene C4, prostaglandin D2, and platelet-activating factor constitutively. This release was not potentiated by immunologic- or nonimmunologic-activating stimuli. In addition, they exhibited cytochemical and surface phenotypic features of monocytes. Our results indicate that a population of abnormal proliferative cells exist in the marrow of this patient; that these cells may be responsible for the patient's pronounced systemic proliferation of mast cells and the associated symptoms; and that the cell's mast cell, monocyte properties may be indicative of a common bone marrow-derived mast cell/monocyte precursor.

Antibodies, Monoclonal↗
PubMed1991

Stage-specific expression of intracisternal A-particle sequences in murine myelomonocytic leukemia cell lines and normal myelomonocytic differentiation.

The levels of intracisternal A-particle (IAP) mRNA were analyzed in a variety of myelomonocytic leukemia cell lines, peritoneally derived macrophages, and normal hemopoietic progenitors induced to differentiate. In both normal and leukemic cells, the highest level of IAP message was found in cells at an intermediate stage of myelomonocytic differentiation, namely, the promyelomonocyte. These results indicate that IAP sequence transcription is regulated differentially during myelomonocytic cell development and that in general, the expression pattern is preserved in leukemic cell lines in vitro. In addition, Northern (RNA) analysis detected only type I IAP transcripts as the major IAP message and the expressed IAP subtypes varied in certain cell lines. This is the first comprehensive study of IAP expression in the myelomonocytic lineage and provides a useful system to study the biology of IAPs.

Animals↗
PubMed1991

Tumor progression following transformation of murine monocytes by v-myc: acquisition of immortalization and tumorigenicity.

Monocyte transformation by the v-myc oncogene has been used to study myelomonocytic tumor progression in vitro. Murine monocytes transformed by a recombinant retrovirus containing MC29 v-myc were found to exhibit a proliferative burst to day 28-40 post-infection. There-after growth slowed and cell number remained relatively static to day 80-90 post-infection. During both the proliferative and quiescent periods, the cells were dependent on the myelomonocytic growth factor CSF-1 for growth and viability. Analysis of this transformation revealed that the initial transformants were polyclonal, non-immortal, and non-tumorigenic in syngeneic mice. At day 80-90 post infection, a fresh round of cellular proliferation occurred and, in contrast to the initial burst, growth was sustained allowing the establishment of cell lines. These lines were found to be monoclonal, immortal, growth factor independent and, in certain cases, tumorigenic in syngeneic mice. Associated with the establishment of growth factor independent cell lines was the constitutive synthesis of the myelomonocytic growth factor, CSF-1. Proto-oncogene screening of the initial transformants and the cell lines also revealed the expression of c-raf and the CSF-1 receptor, c-fms. Our results indicate that, following transformation by v-myc, monocytes can progress in vitro to become growth factor independent and immortal and that both monocyte transformation and immortalization can be dissociated from tumorigenicity.

Animals↗
PubMed1991

Overexpression of the retinoblastoma gene in a familial adrenocortical carcinoma.

Tumors derived from a Li-Fraumeni syndrome cancer-susceptible family were examined for expression of the retinoblastoma susceptibility gene (RB). Whereas RB expression was normal in a primary breast carcinoma and its metastases from one member of this family, overexpression of RB was found in an adrenocortical carcinoma from another family member. This was in contrast to normal RB expression in normal tissue of this patient, the adrenocortical adenocarcinoma cell line SW-13, and the fibroblast cell line MRC-5, and low level RB expression in normal adrenal tissue. The overexpression in the adrenocortical carcinoma resulted in increased synthesis of the RB-encoded protein and did not appear to be associated with RB amplification or rearrangement. This result is novel as it is usually the loss of expression or production of an altered RB transcript exhibiting deletions that is associated with carcinogenesis. In light of the recent discovery of p53 point mutations in the affected Li-Fraumeni syndrome family members tested, RB overexpression may constitute a secondary event in Li-Fraumeni syndrome tumorigenesis.

Adrenal Cortex Neoplasms↗
PubMed1990

Nucleotide sequence and genomic organization of a human T lymphocyte serine protease gene.

We have determined the nucleotide sequence of 4508 base pairs of human genomic DNA which contain the human serine esterase gene from cytotoxic T lymphocytes (SECT) (equivalent to the 1-3E cDNA clone) and include 879 bp of 5' flanking DNA and 393 bp of 3' flanking DNA. The gene consists of five exons of 88, 148, 136, 261, and 257 nucleotides separated by four introns of 1043, 455, 205, and 643 nucleotides. The location of introns with respect to protein coding sequences in the SECT gene is identical to that of the human cathepsin G and murine granzyme B genes. Comparison of SECT gene exonic sequences to murine granzyme B-F cDNA sequences indicates similarities of 75 and 72% for granzymes B and C and 61, 59, and 61% for granzymes D, E, and F, respectively. The 5' flanking sequence of the SECT gene showed similarity only to the 5' flanking sequence of the murine granzyme B gene, indicating that these genes are homologous. Comparison of the SECT gene sequence to the human cathepsin G sequence indicated no similarity in the 5' flanking DNA although the exonic sequences show 64% sequence similarity overall and 45% sequence similarity in the respective 3' untranslated regions. These similarities suggest that the SECT and cathepsin G genes are members of the same family of serine protease genes. Evidence from high and low stringency Southern transfer analysis of human genomic DNA indicates the presence of another gene of at least 85% sequence similarity to the SECT gene.

Base Sequence↗
PubMed1988

Structure and differential mechanisms of regulation of expression of a serine esterase gene in activated human T lymphocytes.

A cDNA clone encoding a polypeptide resembling proteolytic serine esterases (from cytotoxic T-cells; SECT) was isolated from human peripheral blood lymphocytes which had been cultured in the presence of the T-cell mitogen phytohemagglutinin for 72 h. The cDNA encodes a polypeptide of 247 amino acids which show homology of 99% with the protein sequence encoded by a cDNA clone (1-3E) isolated from staphylococcal enterotoxin A-stimulated human peripheral blood lymphocytes and 68% with the protein sequence (cytotoxic cell protease type I) derived from a cDNA clone (C11) encoding a serine esterase isolated from a murine cytotoxic T-cell line. The overall nucleotide sequence homology between the SECT cDNA and 1-3E was 99% and 73% between SECT and C11. Comparing the coding regions of SECT and C11 showed 75% homology, whereas the 5'- and 3'-untranslated regions showed 67% homology. Phytohemagglutinin stimulation results in 30-, 60-, and 370-fold increases in cytoplasmic SECT mRNA with respect to unstimulated cells after 6-, 24-, and 72-h cultures, respectively. At 6 h, the increase in SECT mRNA occurs in the absence of increases in SECT gene transcription and cytoplasmic mRNA stabilization. A 5-fold increase in SECT nuclear RNA seen at this time suggests that stabilization of SECT nuclear RNA transcript is responsible for early increases in SECT mRNA levels. At 24 and 72 h, the increased cytoplasmic SECT mRNA levels can be accounted for by increased transcriptional activity of the SECT gene.

Amino Acid Sequence↗
PubMed1985

5,10-Methylenetetrahydrofolate reductase deficiency. Clinical and biochemical features of a further case.

We report the case of a boy with 5,10-methylenetetrahydrofolate reductase deficiency. The clinical features consisted of severe mental retardation, spasticity and seizures remaining static to 7 years of age followed by a phase of rapid deterioration and death at 7 1/2 years of age. The main biochemical findings were homocystinaemia, homocystinuria, a normal methionine level in plasma and cerebrospinal fluid, an increased excretion of methionine in urine and a very low level of folate in the cerebrospinal fluid. The activity of 5,10-methylenetetrahydrofolate reductase was greatly reduced in the patient's lymphocytes and liver.

5,10-Methylenetetrahydrofolate Reductase (FADH2)↗
PubMed1985

One-carbon metabolism in lectin-activated human lymphocytes.

Serine is an essential amino acid for the lectin-mediated transformation of human peripheral blood lymphocytes due to the inability of this cell to synthesize sufficient quantities via either the phosphorylated pathway or by reversal of the serine hydroxymethyltransferase reaction to meet the metabolic demands. The level of intracellular serine is tightly regulated, and the culture medium concentration for optimum cellular transformation falls within a relatively narrow range. The three-carbon atom of serine is the major source of one-carbon units required for purine and pyrimidine nucleotide biosynthesis, but the key effect of both serine deprivation and of high medium serine levels would appear to be on protein synthesis. Although an alternative source of one-carbon units, as provided by high levels of formate in the culture medium, can partially reverse the effects of serine deprivation, the only other demonstrable source of one-carbon units, tryptophan, requires serine for its incorporation and subsequent metabolism. Methionine is also essential for lymphocyte transformation and is involved in the synthesis of a small amount of phosphatidylcholine, although most of this phospholipid is provided by choline and lysophosphatidylcholine from the serum-supplemented culture medium.

Blood Proteins↗
PubMed1985

Growth-related changes in specific mRNAs upon lectin activation of human lymphocytes.

A cDNA library in lambda gt10 was constructed from the cytoplasmic poly(A) +RNA of human peripheral blood lymphocytes after 72 hr of phytohemagglutinin stimulation, with the aim of assessing selective gene expression as a result of lymphocyte activation. Thirteen recombinants were isolated by the use of an enriched probe and differential screening. These clones were categorized into two groups with respect to their hybridization to mRNA. In the first group three recombinants were isolated, which hybridized to single discrete mRNAs in the size range 0.7-1.7 kb. The mRNAs corresponding to these clones were present at elevated levels in activated lymphocytes, but the kinetics of increase differed. The 0.7-kb mRNA coded for by clone p1L1 increased two-fold at 6 hr and remained elevated over 72 hr, as did beta-actin mRNA. The 1.7-kb mRNA coded for by clone p9L2 increased two- to three-fold after 6 hr and was maximally expressed after 24 hr exposure to phytohemagglutinin, coincident with the onset of DNA replication, and maintained this level up to 72 hr. The 1.0-kb mRNA coded by p10L2F which was rare in resting cells increased 25- to 30-fold after 6 hr, prior to overall transcriptional increases and reached peak levels after 72 hr when a substantial proportion of the cells were in the S and G2 + M phases of the cell cycle. This clone was undetectable or very rare in the leukemic T-lymphoblast cell line CCRF-CEM. The second group of clones, consisting of the remaining 10 recombinants, did not hybridize to discrete bands, but to a smear on RNA blots.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗