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H P Koeffler

Publications and source records attributed to H P Koeffler.

At least 19 recordsLinked to original sources
PubMed1992

Irradiation increases levels of GM-CSF through RNA stabilization which requires an AU-rich region in cancer cells.

Granulocyte/macrophage colony stimulating factor (GM-CSF) is a hematopoietic growth factor that stimulates a wide range of myeloid hematopoietic cells; RNAs coding for many oncogenes and cytokines including GM-CSF have a very short half-life. The motif of AUUUA is a highly conserved sequence in the 3'untranslated regions (3'UTR) of these transcripts and is repeated a number of times in these short-lived cytokines and oncogenes. These sequences play a major role in controlling stability of these transcripts. Human cancer cells were transfected with a chimeric rabbit beta-globin gene linked to either a 58 bp sequence of the AT-rich region from GM-CSF or a control sequence. We have found that irradiation stimulates accumulation of GM-CSF, interleukin-6 (IL-6), and IL-1 beta RNAs. In addition, this accumulation of GM-CSF was at least, in part, a result of increased stabilization of GM-CSF transcripts. Further experiments showed that irradiation increased levels of the chimeric beta-globin transcripts containing AUUUA sequences from GM-CSF, but not those containing the control sequences. Our results suggest that irradiation increases expression of GM-CSF RNA and that posttranscriptional stabilization requiring AUUUA sequences probably is in part one of the mechanisms producing the increased levels of GM-CSF RNA by irradiation.

Base Sequence↗
PubMed1992

Tumor necrosis factor stimulates Na+/H+ antiporter in human fibroblasts: dissociation between intracellular alkalinization and cytokine mRNA accumulation.

Several hormones and cytokines stimulate the cellular Na+/H+ antiporter and this stimulation may be a signal transduction mechanism to mediate gene expression. We find that tumor necrosis factor rapidly stimulates both the Na+/H+ antiporter and the accumulation of mRNA coding for granulocyte-macrophage colony-stimulating factor and interleukin-6 in fibroblasts. Further experiments show that these phenomena occur independent of each other.

Carrier Proteins↗
PubMed1992

p53 mutations in human lung tumors.

Mutation of one p53 allele and loss of the normal p53 allele [loss of heterozygosity (LOH)] occur in many tumors including lung cancers. These alterations apparently contribute to development of cancer by interfering with the tumor suppressor activity of p53. We directly sequenced amplified DNA in the mutational hot spots (exons 4-8) of p53 in DNA samples from 40 lung cancers. Most (31 of 40) samples were preselected for LOH in the region of p53. We detected 23 p53 mutations within these exons in 22 lung cancers; no p53 mutations were found in normal tissue of the patients. One-half of the mutations were G to T transversions on the nontranscribed strand, consistent with mutagenesis by tobacco smoke. Mutations of C to A on the nontranscribed strand, which would result from G to T mutations on the transcribed strand, were detected only in one sample. Three of 23 mutations were nonsense mutations; to date, nonsense mutations of p53 have not been reported in lung cancer. Mutation of this p53-coding region was detected in 20 of 27 small cell lung cancer samples, representing a 70% occurrence. Mutation of the p53 gene is apparently very frequent in small cell lung cancers. When LOH in the p53 region could be determined, complete concordance occurred between a sample having both a p53 mutation and LOH in the region of p53 (18 of 18 samples). Twelve samples of lung cancer had LOH in the region of p53, but the samples had no detectable p53 mutations, suggesting either alterations outside the known mutational hot spots of p53 or alterations of another unidentified tumor suppressor gene in the region of p53.

Amino Acid Sequence↗
PubMed1992

Mutations of the p53 gene in adult T-cell leukemia.

The p53 tumor suppressor gene was examined by direct sequencing of polymerase chain reaction-amplified DNA from fresh tumor cells of 10 patients with adult T-cell leukemia (ATL). Samples included nine patients with acute or lymphomatous ATL, and one patient in whom samples were examined in both his acute and chronic stages of ATL. Four missense mutations and one silent point mutation in the coding region of the p53 gene were found in cells from five patients with either acute or lymphomatous ATL. The missense mutations were homozygous and occurred in evolutionarily highly conserved regions of p53. One patient had no p53 mutation in his leukemic cells during chronic phase of ATL, but had a homozygous point mutation at codon 273 (Arg to His) when he progressed to acute ATL. In summary, we show that p53 is frequently mutated in the acute phase of ATL and one informative case suggests that p53 mutations may be associated with the transition from chronic to acute ATL.

Adult↗
PubMed1992

Differentiation therapy.

A variety of compounds are able to induce myeloid leukemia cells to differentiate into morphologically and functionally mature cells. Vitamin D derivatives, retinoids, interferons, and polar-planar compounds are agents that demonstrate such activity in vitro and have been employed as therapy for the myelodysplastic syndromes.

Calcitriol↗
PubMed1992

Immunohistochemical analysis of p53 expression in malignant lymphomas.

p53 is a tumor suppressor gene that commonly undergoes mutations in human tumors, including lymphomas. Because p53 mutations are not restricted to a single locus, immunohistochemistry is useful to detect p53 expression and correlate this finding with lymphoma phenotype. Cryostat sections from 125 cases of lymphoma were analyzed for p53 expression using three different monoclonal antibodies (pAb 421, 1801, 240) which react with human cellular p53 and a common conformational epitope on mutant p53. A control antibody (pAb 246) reacts only with wild type p53 of murine origin and was negative in all cases. Tissue from 29 cases of lymphoid hyperplasia, including six from human immunodeficiency virus-positive (HIV+) patients, were negative for p53. p53 was predominantly localized in nuclei of high-grade lymphomas, including 14 of 46 cases of B cell immunoblastic lymphomas and two of five T cell immunoblastic lymphomas. p53 expression was relatively common in lymphomas from HIV+ patients, and unusual in intermediate and low-grade lymphomas of follicular center cell type. Low-grade lymphoma of small lymphocytic type disclosed p53+ large cells (paraimmunoblasts) that may play a role in tumor progression in this lymphoma subtype. p53 was also strongly expressed in the nuclei of Reed Sternberg cells from 19 of 37 cases of Hodgkin's disease, including six cases of mixed cellularity, and 13 cases of nodular sclerosing type. Immunohistochemical staining is a rapid method to identify p53 expression in lymphomas.

Animals↗
PubMed1992

Prevalence of N-ras mutations in children with myelodysplastic syndromes and acute myeloid leukemia.

The ras proto-oncogene family encodes a group of 21 kDa nucleotide-binding proteins. Activating mutations of ras genes are associated with certain types of malignancies, indicating that they are related in some way to the malignant process. We have examined bone marrow cells from nine children with myelodysplastic syndromes (MDS) and 35 with acute myeloid leukemia (AML) for activating point mutations of ras genes by in vitro amplification using polymerase chain reaction (PCR), oligonucleotide hybridization and sequencing of PCR products. We found N-ras mutations in cells from 3 of 9 children (33%) with MDS and only 2 of 35 children with AML (6%; 95% confidence interval is 0.7-19%). All mutations the second nucleotide of codon 12 or the first nucleotide of codon 61 of N-ras. There was no apparent correlation with clinical or laboratory characteristics, including karyotype; however, an association of N-ras activation with the most aggressive type of MDS was noted. Among the patients with MDS, 2 of 6 with monosomy 7 had N-ras mutations; however, three children with monosomy 7 which presented with AML lacked ras mutations. One patient was studied at time of diagnosis of MDS and again after progression to AML. At the preleukemic stage of disease, an N-ras mutation was identified; however, after development of AML this mutation was not present in the leukemic clone. In conclusion, these data show that ras mutations, while not necessary for leukemic transformation, may be important for the initiation of preleukemias evolving into overt AML.

Adolescent↗
PubMed1992

Therapy-related myelodysplastic syndromes.

This article summarizes the clinical aspects, cytogenetics, and initiating agents associated with therapy-related myelodysplastic syndromes. Details regarding associated chemotherapeutic agents and ionizing radiation are discussed and therapeutic considerations are presented.

Antineoplastic Agents↗
PubMed1991

Role of AUUU sequences in stabilization of granulocyte-macrophage colony-stimulating factor RNA in stimulated cells.

RNAs for transiently expressed genes such as oncogenes and cytokines, including granulocyte-monocyte colony-stimulating factor (GM-CSF), have a short half-life (T1/2). A cluster of AUUU sequences identified in the 3' untranslated (UT) region of these RNAs has been implicated in controlling stability of these transcripts. We examined the role of AUUU sequences in mRNA stability of GM-CSF after stimulation of cells. Human fibroblasts (W138) were stably transfected with chimeric constructs containing the beta-globin gene linked to a 52-bp tail of GM-CSF containing either eight ATTTT (pNEOR beta G-AT) or eight repeats in which the AT sequences have been changed to GC sequences (pNEOR beta G-GC). Data confirmed that AUUU sequences in 3'UT region of GM-CSF play a major role in GM-CSF RNA instability. Stimulators of protein kinase C (PKC), cycloheximide (CHX), sodium fluoride (NaF), and, to a more limited extent, interleukin-1 beta (IL-1 beta), appear to stabilize GM-CSF RNA through these AUUU sequences, but tumor necrosis factor-alpha (TNF-alpha) induces stabilization of GM-CSF RNA through a mechanism independent of their AUUU sequences.

Base Sequence↗
PubMed1991

Changes of DNA methylation and chromatin structure in the human myeloperoxidase gene during myeloid differentiation.

Expression of the myeloperoxidase (MPO) gene is tightly regulated in a tissue- and development-specific manner. Accumulation of MPO messenger RNA (mRNA) occurs only at the late myeloblastic and promyelocytic stages of myeloid differentiation and is negligible at other stages of myeloid development and in other tissues. The goal of our studies was to begin to understand the events that occur to control MPO gene expression during normal granulocytopoiesis. Chromatin structure of the MPO gene was evaluated by DNase I treatment of isolated nuclei and Southern blot analysis. No detectable DNase I hypersensitive sites were found in the region of the MPO gene in non-myeloid cells. One site was present in the 5' upstream region in myeloid cells that are developmentally too immature to transcribe MPO. Three sites of hypersensitivity in the regions of the putative MPO promoter and upstream region occurred in MPO-expressing promyelocytes. These sites were markedly reduced in terminally differentiated, non-expressing myeloid cells. Analysis of DNA methylation of the MPO gene using methylation-sensitive restriction enzymes showed that the gene was highly methylated in non-myeloid cells. Stepwise demethylation occurred in myeloid cells developmentally too immature to transcribe MPO. Maximal demethylation in the 5' gene region occurred in MPO-expressing promyelocytes. This methylation pattern did not change in terminally differentiated, MPO non-expressing myeloid cells. A somatic hybrid cell formed by fusion of HL-60 (MPO-expressing cells) and PUT (MPO non-expressing lymphoid cells) extinguished expression of MPO and showed a chimeric pattern of MPO gene methylation, suggesting that demethylation is necessary but not sufficient for expression of the MPO gene. Our studies show that demethylation and DNase I hypersensitivity of the MPO gene were associated with a tissue-dependent potential for MPO gene expression that preceded the developmental ability to express MPO mRNA.

Base Sequence↗
PubMed1991

Development of a novel 1,25(OH)2-vitamin D3 analog with potent ability to induce HL-60 cell differentiation without modulating calcium metabolism.

We describe several novel analogs of the seco-steroid 1,25(OH)2-vitamin D3[1,25(OH)2D3] and their effects on differentiation and proliferation of HL-60 human myeloid leukemic cells in vitro as well as their effects on calcium metabolism in vivo. The 1 alpha-25(OH)2-16ene-23yne-26,27F6-vitamin D3 is the most potent analog reported to date, having about 80-fold more activity than the reference 1,25(OH)2D3 for inhibition of proliferation and induction of differentiation of HL-60 cells. Also, this analog decreased RNA expression of MYC oncogene in HL-60 by 90% at 5 x 10(-10) mol/L. Intriguingly, intestinal calcium absorption and bone calcium mobilization mediated in vivo by 1 alpha-25(OH)2-16ene-23yne-26,27F6-D3 was found to be markedly (15-fold) less than that of 1,25(OH)2D3. In addition, 1 alpha-25(OH)2D3 bound to 1,25(OH)2D3 receptors of both HL-60 and intestine more avidly than did 1 alpha-25(OH)2-16ene-23yne-26,27F6-D3. This novel analog may open up new therapeutic strategies for several hematopoietic, skin, and bone abnormalities and may provide a new tool to understand how vitamin D3 seco-steroids induce cellular differentiation.

Bone Marrow↗
PubMed1991

Cytokine messenger RNA stability is enhanced in tumor cells.

Hematopoietic growth factors are produced by a number of human tumors. We extracted RNA from selected human tumor cells known to produce at least one hematopoietic growth factor and found high levels of abnormally stable cytokine messenger (m)RNA. Half-life experiments performed after preventing RNA synthesis by exposing cells to actinomycin D before RNA extraction showed stabilization of cytokine messages in tumor cells in liquid culture as well as in human tumor xenografts grown in mice. Exposure to the phorbol ester phorbol 12-myristate 13-acetate (TPA) caused enhancement of granulocyte-macrophage colony-stimulating factor (GM-CSF) message level in lung cancer cells and in control fibroblasts but elevated levels persisted far longer in the tumor cells. In normal cells, an AU-rich sequence in the 3' untranslated region of cytokine mRNAs confers lability to the message. Although a beta-globin gene expression vector containing this region appears to produce unstable mRNA in lung cancer cells, cytokine mRNAs, which also contain this sequence, are very stable in the tumors we studied. This may indicate that another region of the cytokine mRNA molecule is of greater importance than the AU-rich region in determining mRNA stability in tumor cells.

Adenocarcinoma↗
PubMed1991

1,25-Dihydroxyvitamin D3 receptor RNA: expression in hematopoietic cells.

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] induces differentiation and inhibits proliferation of myeloid leukemic cells from various lines and patients; these effects are probably mediated through the 1,25(OH)2D3 receptor. Little is known of expression of 1,25(OH)2D3 receptor RNA in hematopoietic cells. We examined the expression and modulation of expression of 1,25(OH)2D3 receptor RNA in various proliferating and nonproliferating hematopoietic cells. Constitutive expression of 1,25(OH)2D3 receptor RNA was detected in various kinds of hematopoietic cells, including macrophages and activated T lymphocytes, as well as in cell lines KG-1 (myeloblasts), HL-60 (promyelocytes), ML-3 (myelomonoblasts), U937, THP-1 (monoblasts), K562 (erythroblasts), and S-LB1 (HTLV-1-transfected T lymphocytes). Receptor transcripts were 4.6 kilobases (kb), and no variant sizes were observed. All cell lines examined in this group also expressed 1,25(OH)2D3 receptors. Most B lymphocyte lines expressed negligible levels of 1,25(OH)2D3 receptor RNA and protein; however; analysis of a lymphoid/myeloid somatic hybrid suggested that suppression of expression of 1,25(OH)2D3 receptor RNA in B lymphocytes may be a dominant characteristic. HL-60 cells were cultured with 10(-7) mol/L 1,25(OH)2D3 for 24 to 72 hours, and levels of expression of 1,25(OH)2D3 receptor and its RNA were examined. Levels of RNA coding for the receptor were not modulated by exposure to high levels of ligand. Levels of occupied 1,25(OH)2D3 receptor protein increased in these HL-60 cells; but the total number of 1,25(OH)2D3 receptors decreased about 50% at 24 hours and returned toward normal at 72 hours. Steady-state levels of 1,25(OH)2D3 receptor RNA were not affected by terminal differentiation of HL-60 toward either granulocytes or macrophages. Nondividing macrophages from normal individuals also expressed 1,25(OH)2D3 receptor RNA. In contrast, nondividing peripheral blood lymphocytes from normal individuals did not express 1,25(OH)2D3 receptor RNA; with stimulation of proliferation of these cells, accumulation of 1,25(OH)2D3 receptor RNA increased markedly. Half-life (t1/2) of 1,25(OH)2D3 receptor RNA in T lymphocytes was short (1 hour) as determined by measuring decay of the message after addition of actinomycin D. Consistent with this short t1/2, accumulation of 1,25(OH)2D3 receptor RNA increased in cells as their protein synthesis was inhibited. Further studies are required to understand the physiologic role of 1,25(OH)2D3 receptors in myeloid cells and proliferating T lymphocytes.

Calcitriol↗
PubMed1991

Expression of hematopoietic growth factor RNAs in human mesenchymal cells from various organs.

Experiments were undertaken to study expression of hematopoietic growth factor RNAs in mesenchymal cells from a variety of organs including bone marrow, foreskin, gingiva, and lung. Cells from each organ had negligible expression of RNAs coding for granulocyte (G), macrophage (M), and granulocyte-macrophage (GM) colony stimulating factor (CSF), interleukin 1 beta (IL-1 beta), and IL-6. Fibroblasts from each tissue had a comparable ability to express the same cytokine RNAs. Surprisingly, the stimuli for expression of G-CSF RNA was disparate from the stimuli for expression of the other cytokine RNAs. While IL-1 beta enhanced accumulation of G-CSF RNA, tumor necrosis factor alpha (TNF) and 12-O-tetradecanoylphorbol-13-acetate (TPA) did not. In contrast, IL-1 beta, TNF, and TPA equally stimulated increased levels of M-CSF, GM-CSF, IL-1 beta and IL-6 RNAs.

Bone Marrow↗
PubMed1991

D-factor: modulation of expression in fibroblasts.

Differentiation inducing factor (D-factor) is a recently described protein. The gene has been cloned, but little is known concerning regulation of expression of the gene. Our study showed that fibroblasts from a variety of tissues (lung, bone marrow, gingiva, foreskin) constitutively expressed D-factor RNA. Levels of expression of this gene increased in fibroblasts of each of the tissues after exposure to several stimuli including products of activated macrophages and lymphocytes (tumor necrosis factor (TNF), interleukin 1 and lymphotoxin). Other stimuli were those capable of activating either protein kinase C (12-O-tetradecanoylphorbol-13-acetate (TPA) and teleocidin), G-binding proteins (NaF) or those inhibiting protein synthesis (cycloheximide). Accumulation of D-factor RNA by TNF may in part be explained by stabilization of D-factor transcripts; TPA and cycloheximide clearly stabilized D-factor transcripts. We and others have shown that these same signals similarly stimulated fibroblasts to express RNAs coding for a variety of cytokines including three colony-stimulating factors as well as interleukins 1 and 6. Taken together, D-factor probably is a participant in the cascade of cytokines that are produced in mesenchymal cells after various stimuli such as bacterial invasion.

Cells, Cultured↗
PubMed1991

Evidence for 1,25-dihydroxyvitamin D3 production by cultured porcine alveolar macrophages.

Previous studies have demonstrated that human alveolar and bone marrow macrophages when activated in vitro can metabolize 25-hydroxyvitamin[3H]D3 to 1 alpha,25-dihydroxyvitamin[3H]D3; however, to date no animal models to study this system have been available. In the present study, cultured porcine pulmonary alveolar macrophages from two animals were assayed for their capability for metabolism of 25-hydroxyvitamin[3H]D3. The porcine alveolar macrophages constitutively produced a metabolite of 25-hydroxyvitamin[3H]D3 which was identified as 1 alpha,25-dihydroxyvitamin[3H]D3 by high performance liquid chromatography. The apparent KM was in the range of 300 nM. Unlike human macrophages, treatment of porcine alveolar macrophages with lipopolysaccharide did not stimulate 1 alpha,25-dihydroxyvitamin[3H]D3 production. Addition of 1 alpha,25-dihydroxyvitamin-D3 to macrophages cultures led to a sensitive proportional inhibition of 1 alpha,25-dihydroxyvitamin[3H]D3 synthesis.

Animals↗