Biomedical subjects

X Wu

Publications and source records attributed to X Wu.

At least 667 records · Page 37Linked to original sources

PubMed1997

Genes for Tfb2, Tfb3, and Tfb4 subunits of yeast transcription/repair factor IIH. Homology to human cyclin-dependent kinase activating kinase and IIH subunits.

Genes for the Tfb2, Tfb3, and Tfb4 subunits of yeast RNA polymerase transcription factor IIH (TFIIH) are described. All three genes are essential for cell viability, and antibodies against Tfb3 specifically inhibit transcription in vitro. A C-terminal deletion of Tfb2 caused a defect in nucleotide excision repair, as shown by UV sensitivity of the mutant strain and loss of nucleotide excision repair activity in cell extracts (restored by the addition of purified TFIIH). An interaction between Tfb3 and the Kin28 subunit of TFIIH was detected by the two-hybrid approach, consistent with a role for Tfb3 in linking kinase and core domains of the factor. The deduced amino acid sequence of Tfb2 is similar to that of the 52-kDa subunit of human TFIIH, while Tfb3 is identified as a RING finger protein homologous to the 36-kDa subunit of murine CAK (cyclin-dependent kinase activating kinase) and to the 32-kDa subunit of human TFIIH. Tfb4 is homologous to p34 of human TFIIH and is identified as the weakly associated 37-kDa subunit of the yeast factor. These and other findings reveal a one-to-one correspondence and high degree of sequence similarity between the entire set of yeast and human TFIIH polypeptides.

Amino Acid Sequence↗

PubMed1997

Activity of DNA ligase IV stimulated by complex formation with XRCC4 protein in mammalian cells.

Mutation of the XRCC4 gene in mammalian cells prevents the formation of the signal and coding joints in the V(D)J recombination reaction, which is necessary for production of a functional immunoglobulin gene, and renders the cells highly sensitive to ionizing radiation. However, XRCC4 shares no sequence homology with other proteins, nor does it have a biochemical activity to indicate what its function might be. Here we show that DNA ligase IV co-immunoprecipitates with XRCC4 and that these two proteins specifically interact with one another in a yeast two-hybrid system. Ligation of DNA double-strand breaks in a cell-free system by DNA ligase IV is increased fivefold by purified XRCC4 and seven- to eightfold when XRCC4 is co-expressed with DNA ligase IV. We conclude that the biological consequences of mutating XRCC4 are primarily due to the loss of its stimulatory effect on DNA ligase IV: the function of the XRCC4-DNA ligase IV complex may be to carry out the final steps of V(D)J recombination and joining of DNA ends.

Animals↗

PubMed1997

Regulatory volume decrease by SV40-transformed rabbit corneal epithelial cells requires ryanodine-sensitive Ca2+-induced Ca2+ release.

The relationship between relative cell volume and time-dependent changes in intracellular Ca2+ concentration ([Ca2+]i) during exposure to hypotonicity was characterized in SV-40 transformed rabbit corneal epithelial cells (tRCE) (i). Light scattering measurements revealed rapid initial swelling with subsequent 97% recovery of relative cell volume (characteristic time (tauvr) was 5.9 min); (ii). Fura2-fluorescence single-cell imaging showed that [Ca2+]i initially rose by 216% in 30 sec with subsequent return to near baseline level after another 100 sec. Both relative cell volume recovery and [Ca2+]i transients were inhibited by either: (a) Ca2+-free medium; (b) 5 mM Ni2+ (inhibitor of plasmalemma Ca2+ influx); (c) 10 microM cyclopiazonic acid, CPA (which causes depletion of intracellular Ca2+ content); or (d) 100 microM ryanodine (inhibitor of Ca2+ release from intracellular stores). To determine the temporal relationship between an increased plasmalemma Ca2+ influx and the emptying of intracellular Ca2+ stores during the [Ca2+]i transients, Mn2+ quenching of fura2-fluorescence was quantified. In the presence of CPA, hypotonic challenge increased plasmalemma Mn2+ permeability 6-fold. However, Mn2+ permeability remained unchanged during exposure to either: 1.100 microM ryanodine; 2.10 microM CPA and 100 microM ryanodine. This report for the first time documents the time dependence of the components of the [Ca2+]i transient required for a regulatory volume decrease (RVD). The results show that ryanodine sensitive Ca2+ release from an intracellular store leads to a subsequent increase in plasmalemma Ca2+ influx, and that both are required for cells to undergo RVD.

Animals↗

PubMed1997

Dose-volume histograms for bladder and rectum.

PURPOSE: A careful examination of the foundation upon which the concept of the Dose-Volume Histogram (DVH) is built, and the implications of this set of parameters on the clinical application and interpretation of the DVH concept has not been conducted since the introduction of DVHs as a tool for the quantitative evaluation of treatment plans. The purpose of the work presented herein is to illustrate problems with current methods of implementing and interpreting DVHs when applied to hollow anatomic structures such as the bladder and rectum. METHODS AND MATERIALS: A typical treatment plan for external beam irradiation of a patient with prostate cancer was chosen to provide a data set from which DVH curves for both the bladder and rectum were calculated. The two organs share the property of being shells with contents that are of no clinical importance. DVHs for both organs were computed using a solid model and using a shell model. Typical treatment plans for prostate cancer were used to generate DVH curves for both models. The Normal Tissue Complication Probability (NTCP) for these organs is discussed in this context. RESULTS: For an eight-field conformal treatment plan of the prostate, a bladder DVH curve generated using the shell model is higher than the corresponding curve generated using the solid model. The shell model also has a higher NTCP. A six-field conformal treatment plan also results in a higher DVH curve for the shell model. A treatment plan consisting of bilateral 120-degree arcs, results in a higher DVH curve for the shell model, as well as a higher NTCP. CONCLUSION: The DVH concept currently used in evaluation of treatment plans is problematic because current practices of defining exactly what constitutes "bladder" and "rectum." Commonly used methods of tracing the bladder and rectum imply use of a solid structure model for DVHs. In reality, these organs are shells and the critical structure associated with NTCP is obviously and indisputably the shell, as opposed to its contents. Treatment planning algorithms for DVH computation should thus be modified to utilize the shell model for these organs.

Humans↗

PubMed1997

Structure/calcium affinity relationships of site III of calmodulin: testing the acid pair hypothesis using calmodulin mutants.

Calmodulin mutants in which the calcium binding affinity of site IV was greatly reduced by a D133E mutation were prepared using site-specific, cassette-mediated mutagenesis as a multisite calcium binding protein model to examine structure/calcium affinity relationships in site III of calmodulin. Tryptophan was introduced in position 92 of the calmodulin mutants as a fluorescent label to monitor the calcium-induced structural changes in the C-terminal domain of calmodulin. The five calmodulin mutants, 3xCaM, 3zCaM, 4xCaM, 4zCaM, and 4xzCaM, were designed so that there were three or four acidic amino acid residues in chelating positions of site III with acid pairs on either the X and/or Z coordinating axes. The calcium dissociation constant of site III, KIII, of the five calmodulin mutants changes in a descending order from 3xCaM (237 microM), 3zCaM (140 microM), 4xCaM (5.8 microM), 4zCaM (3 microM), to 4xzCaM (2 microM), and these KIII values are significantly lower than that of F92W/D133E calmodulin (335 microM) in which three acidic residues with no acid pairs were present in site III [Wu, X., & Reid, R. E. (1997) Biochemistry 36, 3608-3616]. These results indicate that the calcium affinity of site III increases when the number of the acidic chelating residues increases from three to four, when the number of acid pairs increases from zero to one and further to two, and when the location of the acid pair is changed from the X axis to the Z axis. This study provides the first evidence that the acid pair hypothesis which correlates the nature of the chelating residues with the calcium affinity of the hlh motif is applicable to a multisite calcium binding protein model. The Hill coefficients indicate that reversal of the sequence of filling of the calcium binding sites in the C-terminal domain from IV --> III to III --> IV also changes the site cooperativity from positive to negative. The cooperativity returns to positive when the proteins are titrated in the presence of a calmodulin-binding peptide. Data from the present study also demonstrate that calmodulin mutants with a decreased calcium affinity have a reduced efficiency in phosphodiesterase regulation at low calcium concentrations (50 microM). However, high calcium concentrations (15 mM) restore the phosphodiesterase regulatory activity of the calmodulin mutants to a level obtained with F92W calmodulin, indicating that the mutations alter calcium regulation of calmodulin-mediated phosphodiesterase activity without affecting the interaction between calmodulin and the enzyme.

Binding Sites↗

PubMed1997

Trigonelline, a naturally occurring constituent of green coffee beans behind the mutagenic activity of roasted coffee?

Trigonelline and amino acids are natural components in green coffee beans. Model systems mimicking coffee roasting were used to produce heated samples of trigonelline, amino acids and glucose. Trigonelline and amino acids were heated separately or in combinations for 20 min at 250 degrees C. The results of bacteria mutation assays (Salmonella typhimurium strains TA 98, YG 1024 and YG 1029) showed that trigonelline, alone or in combination with most of the single amino acids and mixtures of amino acids, yielded potent mutagenic activity. Of the singly heated compounds, the highest mutagenic activity was found for trigonelline. The mutagenic activity detected with metabolic activation of the heated trigonelline samples indicated that the mutagenic compounds might be amines; however, higher mutagenic activity was found for trigonelline and its combinations without metabolic activation, which suggests that other types of mutagens (direct-acting) were predominant. High-performance liquid chromatographic analysis of some of the heated samples did not reveal the presence of any known mutagenic heterocyclic amine.

Alkaloids↗

PubMed1997

Biological monitoring of cadmium exposure and renal effects in a population group residing in a polluted area in China.

In an area of China, not previously studied in detail concerning cadmium pollution and possible adverse effects on the kidney of exposed populations, concentrations of cadmium in urine as an indicator of renal accumulation of cadmium was studied and related to indicators of renal dysfunction in order to examine if a relationship could be documented. Cadmium concentrations in urine were analysed by graphite furnace atomic absorption spectrometry and urinary beta-2 microglobulin (UBM) and albumin (UALB) were measured as indicators of renal dysfunction, Rice samples and urine samples were obtained from three areas in Zhejiang province, China, representing a highly exposed area, a medium exposed area and a control area, respectively. Cadmium concentrations in rice were 3.70, 0.51 and 0.072 mg/kg for the heavily, medium polluted areas and the control area, respectively. Cadmium concentrations in urine (geometric means) were 10.7, 1.62 and 0.40 micrograms/l in the high, medium and control areas respectively. There was a clear increase in UBM and UALB in the heavily exposed group in comparison to the control group and a slight increase in the medium exposed group. There was a statistically significant dose-response relationship between cadmium in urine and beta 2-microglobulin excretion in urine, which is similar to what has previously been reported in other countries. The findings constitute the first report concerning a dose-response relationship in this population group in Zhejiang province in China.

Adult↗

PubMed1997

Acute hematopoietic toxicity of aniline in rats.

In the present study, acute hematopoietic toxicity of aniline as a function of time was investigated in rats. The animals were given a single oral dose of aniline hydrochloride (2 mmol/kg) and euthanized at zero (control), 0.25, 0.5, 1, 3, 6, 12, 24 and 48 h following the treatment. The blood methemoglobin level increased dramatically and attained a peak level of 37% (31 fold greater than the controls) at 0.5 h. Thereafter, the increases were less pronounced and the level declined with time. Spleen weight to body weight ratio remained unchanged up to 24 h, but increased approximately 25% at 48 h. Lipid peroxidation (MDA content) in the spleen increased by 39% at 24 h and remained steady even at 48 h. MDA-protein adducts, as quantitated by a competitive ELISA, showed 94, 126 and 265% increases in the spleen homogenates at 12, 24 and 48 h, respectively, following the treatment. However, no changes were observed in the splenic protein oxidation. Morphological examination showed congestion of splenic blood vessels and marked expansion of red pulp at 24 and 48 h. These studies suggest that aniline related changes in the blood are reflected very early as evident from increases in the methemoglobin content, whereas changes in the spleen appear later and are characterized by splenic weight changes, increased lipid peroxidation, MDA-protein adduct formation and morphological changes after a single high dose exposure. The increased lipid peroxidation in the spleen also suggests that free radical-mediated reactions could be the potential mechanism of splenic toxicity of aniline and lipid peroxidation precedes protein oxidation.

Administration, Oral↗

PubMed1997

A two-site model for ApoB degradation in HepG2 cells.

Newly synthesized apolipoprotein B (apoB) undergoes rapid degradation in a pre-Golgi compartment in HepG2 cells. A major site of this early degradation seems to be on the cytosolic side of the endoplasmic reticulum (ER) membrane and is sensitive to N-acetyl-leucinyl-leucinyl-norleucinal (ALLN), which can inhibit neutral cysteine proteases and/or proteasome activity. Oleate (OA) treatment, which facilitates translocation of nascent apoB across the ER membrane, also reduces early degradation. In the present studies, we have used brefeldin A (BFA), which inhibits vesicular transport from the ER to the Golgi, to demonstrate that apoB can also be degraded by an ER luminal proteolytic activity that is distinct from the ALLN-sensitive proteases. Thus, when BFA-treated HepG2 cells were co-treated with ALLN, which protects apoB but does not facilitate its translocation into the ER lumen, degradation of newly synthesized apoB was significantly reduced compared with cells incubated with BFA alone. However, apoB degradation was rapid and complete when OA was added to media containing either BFA or ALLN/BFA. These results suggested that OA, by increasing translocation of nascent apoB into the ER lumen, exposed apoB to an ALLN-resistant proteolytic pathway. When we incubated HepG2 cells with dithiothreitol (DTT)/OA/BFA or DTT/OA/ALLN/BFA, degradation of apoB was inhibited. Furthermore, addition of DTT resulted in the accumulation of a 70-kDa amino-terminal fragment of apoB. Both full-length and amino-terminal apoB were degraded if DTT was removed from the incubation media; both were secreted if only BFA was removed. Thus, even after apoB is translocated into the ER lumen (thereby avoiding the initial proteolytic pathway), it can potentially be degraded by a lumenal proteolytic process that is ALLN-resistant but DTT-sensitive. The present results, together with previous studies, suggest that at least two distinct steps may be involved in the posttranslational degradation of apoB: 1) the first occurs while apoB is partially translocated and is ALLN-sensitive; and 2) the second occurs in the ER lumen and is DTT-sensitive. Finally, our results support the hypothesis that degradation of partially translocated apoB generates a 70-kDa amino-terminal fragment that is mainly degraded in the ER lumen by a DTT-sensitive pathway.

Apolipoproteins B↗

PubMed1997

Lung carcinoma patients with a family history of cancer and lymphocyte primary chromosome 9 aberrations.

BACKGROUND: Deletion of chromosome 9p has been reported in numerous tumor types. The authors demonstrated in an earlier study that spontaneous chromosome aberrations on chromosome 9 in peripheral blood lymphocytes (PBLs) were a significant risk predictor for lung carcinoma. METHODS: The current study evaluated the relationship between self-reported family history of cancer and spontaneous chromosome aberrations in the PBLs of 97 previously untreated lung carcinoma patients. The authors' hypothesis was that individuals exhibiting specific chromosome aberrations might have inherited genetic instability and thus be more likely to report a family history of cancer. For each individual, a personal interview was conducted to construct a detailed family history, and 100 metaphases from PBLs were analyzed for spontaneous aberrations by G-banding. RESULTS: The patients reported having 829 first-degree relatives, including 74 (8.9%) with cancer. A significantly elevated odds ratio (OR) of 2.7 was noted in smokers for having chromosome 9 aberrations and a first-degree relative with cancer. When the family history of cancer was dichotomized into lung carcinoma or other cancers, the OR associated with chromosomal aberrations was 8.5 for lung carcinoma but only 2.3 for other cancers. In addition to chromosome 9 aberrations, other spontaneous chromosome aberrations and family history of cancer were also evaluated, but no associations were found. There were no associations between age, gender, ethnicity, or smoking status and the chromosome 9 aberration profile. CONCLUSIONS: The findings of this study suggest that chromosome 9 aberrations may be a marker of cancer susceptibility and may be associated with familial aggregation of cancer.

Black People↗

PubMed1997

Chemokines are expressed in a myeloid cell-dependent fashion and mediate distinct functions in immune complex glomerulonephritis in rat.

Using anti-glomerular basement membrane nephritis in rats, we investigated the mechanisms underlying in situ chemokine expression and the in vivo function of these cytokines during the acute phase of this model. We observed that CXC chemokine expression was monophasic and paralleled neutrophil (PMN) influx, whereas CC chemokine expression was biphasic with peaks coinciding with the influx of PMNs and macrophages (Mphi). The initial peak of chemokine expression was attenuated by decomplementation, neutropenia, and leukopenia, while the latter peak was attenuated only by leukopenia and augmented in the accelerated form of this disease model, corresponding to an increase in Mphi influx. Differential expression of chemokines by PMNs and Mphi was not an intrinsic property of these cells, as these leukocytes expressed similar profiles of chemokines in vitro. Immunostaining for Mphi inflammatory protein-1alpha, a CC chemokine, in acute nephritis validated that expression during acute nephritis was accompanied by local protein production. Moreover, neutralizing Ab to Mphi inflammatory protein-1alpha attenuated the acute phase proteinuria, but not the accompanying influx of PMNs. Neutralizing Ab to cytokine-induced neutrophil chemoattractant (a CXC chemokine), in comparison, inhibited both PMN influx and proteinuria. A combination of both Abs was not significantly more effective than either alone. In sum, the influx of myeloid cells is necessary for local chemokine expression in anti-glomerular basement membrane nephritis, although the differential expression of CXC and CC chemokines must involve additional factors. CXC and CC chemokines also mediate distinct, but overlapping, pathophysiologic roles in the acute phase of this model.

Acute-Phase Reaction↗

PubMed1997

Conservative D133E mutation of calmodulin site IV drastically alters calcium binding and phosphodiesterase regulation.

Two calmodulin mutants, F92W and F92W/D133E, were prepared using site-specific cassette-mediated mutagenesis to examine the structure/calcium affinity relationships of cation chelating residues in calcium binding sites III and IV. The mutant, F92W, was prepared to produce a strong fluorescent label to follow the calcium-induced structural changes in the C-terminal domain of the protein. A second mutant, F92W/D133E, was prepared to destroy the calcium binding to site IV and thereby eliminate cooperativity between sites III and IV. The macroscopic calcium dissociation constants of the two sites in the C-terminal domain were derived from the calcium titration data that had been fitted to a two-site Hill equation. The calcium dissociation constants of site III and site IV in the F92W/D133E mutant were 335 microM and 2.76 mM, respectively. These values were significantly greater than the values of 14 and 1 microM for site III and site IV in F92W calmodulin, respectively. These results suggested that a very conservative D133E mutation in the +Z position of the site IV Ca2+-binding loop drastically decreased the calcium binding affinity of the site (2760-fold) and also significantly reduced that of site III in the same domain (24-fold). The D/E calmodulin mutant also had a 3-fold lower phosphodiesterase activation activity with a 25-fold lower affinity for this enzyme than that of F92W calmodulin in the presence of low calcium concentration (50 microM). However, the maximum phosphodiesterase activation activity of the F92W/D133E mutant and the affinity of this mutant for the enzyme were similar to those of F92W calmodulin in the presence of high calcium concentration (15 mM), suggesting that the D133E mutation altered calcium regulation of calmodulin mediated phosphodiesterase activity without affecting calmodulin interaction with the enzyme.

Amino Acid Sequence↗

PubMed1997

Evaluation of beta-globin gene therapy constructs in single copy transgenic mice.

Effective gene therapy constructs based on retrovirus or adeno-associated virus vectors will require regulatory elements that direct expression of genes transduced at single copy. Most beta-globin constructs designed for therapy of beta-thalassemias are regulated by the 5'HS2 component of the locus control region (LCR). Here we show that a human beta-globin gene flanked by two small 5'HS2 core elements or flanked by a 5'HS3 (footprints 1-3) core and a 5'HS2 core are not reproducibly expressed in single copy transgenic mice. In addition, low copy transgene concatamers that contain only dimer 5'HS2 cores fail to express, whereas those that contain monomer 5'HS2 cores express at 14% per copy. These data suggest that spacing between HS cores is crucial for LCR activity. We therefore constructed a novel 3.0 kb LCR cassette in which the 5'HS2, 5'HS3 and 5'HS4 cores are each separated by approximately 700 bp. When linked to the 815 bp beta-globin promoter this LCR directs 45% levels of expression from four independent single copy transgenes. However, the 3.0 kb LCR linked to the 265 bp promoter expresses variable levels, averaging 18%, from three single copy transgenes. Our findings suggest that sequences in the distal promoter play a role in single copy transgene activation and that larger LCR and promoter elements are most suitable for gene therapy applications.

Animals↗

PubMed1997

Cloning and expression of a rat brain interleukin-1beta-converting enzyme (ICE)-related protease (IRP) and its possible role in apoptosis of cultured cerebellar granule neurons.

Several members of the IL-1beta-converting enzyme (ICE) family of proteases recently have been implicated in the intracellular cascade mediating the apoptotic death of various cell types. It is unclear, however, whether ICE-related proteases are involved in apoptosis of mammalian neurons and, if so, how they are activated. Here we report the cloning of an ICE-related protease (IRP) from rat brain, which displays strong sequence identity to human CPP32. In situ hybridization histochemistry reveals that this IRP mRNA is expressed in neuron-enriched regions of the developing and adult rat brain but is profoundly downregulated in the adult (compared with developing) brain. To investigate whether this IRP is involved in the death of neurons in the developing brain, we studied IRP expression in cultured cerebellar granule neurons. In cultured cerebellar granule neurons, reduction of extracellular K+ reliably induces apoptosis and stimulates overexpression of IRP mRNA. The latter is especially prominent 4 hr after switching from high K+ to low K+ medium. The expression of IRP mRNA was maintained at this level for at least 8 hr and was followed by apoptotic death of these neurons. Induction of IRP mRNA and cell death are blocked completely by adding depolarizing concentrations of K+ </=90 min after switching to low K+ medium (i.e., before the commitment point for apoptosis) and partially blocked by brain-derived neurotrophic factor (BDNF), which also partially rescues granule neurons from low K+-induced apoptosis. In addition, overexpression of IRP cDNA in HeLa cells results in cell death accompanied by strong internucleosomal cleavage of DNA, a typical feature of apoptosis. Finally, we detected cleavage of the putative death substrate poly (ADP-ribose) polymerase (PARP), beginning 8 hr after changing from high K+ to low K+ medium, coinciding with the time course of induced expression of the IRP gene. Our data suggest that transcriptional activation of IRP could be one of the mechanisms involved in the apoptotic death of cerebellar granule neurons.

Amino Acid Sequence↗

PubMed1997

Cannabinoids excite dopamine neurons in the ventral tegmentum and substantia nigra.

Extracellular recordings were used to determine the effects of cannabinoids on the activity of dopamine neurons within the ventral tegmental area (VTA) and substantia nigra pars compacta (SNC). Systemic administration of the natural psychoactive cannabinoid delta 9-tetrahydrocannabinol (delta 9-THC) and the synthetic cannabimimetic aminoalkylindole WIN 55,212-2 produced dose-dependent increases in firing rate and burst firing in both neuronal populations. These effects appear to be specific as the non-psychoactive cannabidiol and the inactive enantiomer WIN 55,212-3 failed to alter either parameter of neuronal excitability. Furthermore, dopamine neurons in the VTA were more sensitive than those in the SNC to the stimulatory actions of delta 9-THC. These results may provide a mechanism by which psychoactive cannabinoids increase extracellular dopamine levels in mesolimbic and striatal tissues, and thereby contribute to the reinforcing effects of marijuana.

Analgesics↗

PubMed1997

Characterization of a unique OpMNPV-specific early gene not required for viral infection in tissue culture.

opep-2 is an Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) early gene in the ie1-ie2 gene region for which there is no homolog in either the archetype virus, Autographa californica MNPV, or Bombyx mori NPV. opep-2 is transcribed immediately upon infection as three mRNAs which initiate from a early gene motif (TATA-N27-CAGT). The expression of multiple transcripts at very early times postinfection has only been previously described for the baculovirus early gene ie1, which produces spliced mRNAs. However, distinct from ie1, the multiple mRNAs of opep-2 are due to multiple termination sites and not splicing. Western blot analysis of steady-state levels of OPEP-2 showed that in OpMNPV-infected Ld652Y cells maximum levels are obtained at 8-12 hr postinfection (p.i.) prior to DNA replication. By 48 hr p.i. OPEP-2 is shut off and is undetectable. To aid in elucidating the function of this OpMNPV-specific gene an opep-2 deletion mutant was generated and was compared to wild-type virus to determine if its absence affects viral growth in Ld652Y tissue culture cells.

Amino Acid Sequence↗

PubMed1997

Characterization of a hairy cell leukemia-associated 5q13.3 inversion breakpoint.

Previous cytogenetic analysis has indicated that chromosome anomalies involving the 5q13 band are common in hairy cell leukemia (HCL), occurring in approximately 1/3 of the patients. The data suggest that 5q13.3 is likely to harbor a gene involved in the transformational event of this disease. We selected a constitutional inv(5)(p13.1q13.3) in a patient with HCL as the starting point in an attempt to identify the relevant gene in 5q13.3. By using double color interphase fluorescence in situ hybridization (FISH) techniques, we have identified two cosmid probes from a chromosome 5-specific library that flank the 5q13.3 inversion breakpoint proximally and distally. Pulsed field gel electrophoresis (PFGE) and interphase FISH experiments suggest that the two markers are at a distance of no more than 300 kb. YAC probes covering a 21 Mb region at 5q13 were used to map the 5q13.3 inversion breakpoint and the breakpoint is located within the D5S646-D5S620 region. Two non-chimeric YACs have been identified that span the breakpoint. FISH analysis revealed that four other patients with cytogenetic aberrations of 5q carried inversions/deletions that involved the same 5q13.3 breakpoint region. The identification of a gene involved in hairy cell leukemogenesis in this region will be of major importance in the elucidation of the transformational events of HCL.

Chromosome Deletion↗

PubMed1997

Changes of phosphatidylcholine-specific phospholipase C in hepatocarcinogenesis and in the proliferation and differentiation of rat liver cancer cells.

The biological significance of phosphatidylcholine-specific phospholipase C (PC-PLC) in hepatocarcinogenesis and the proliferation and differentiation of rat liver cancer cells was investigated. The Ca2+-dependent activities of PC-PLC gradually increased during N-nitrosodiethylamine (DEN)-induced hepatocarcinogenesis and peaked at weeks 18-20 when the tumour formed. There was a close relationship between Ca2+-dependent PC-PLC activities and cellular DNA content, membranous gamma-glutamyltranspeptidase (gamma-GT), and tyrosine protein kinase. In contrast, Ca2+-independent PC-PLC activities decreased during hepatocarcinogenesis. Similarly, when CBRH-7919 rat liver cancer cells were treated with phorbol 12-myristate 13-acetate, a proliferation stimulator of the cells, gamma-GT and Ca2+-dependent activities of PC-PLC and the expression of alpha-fetoprotein increased significantly. However, when these cells were induced by retinoic acid to differentiate, Ca2+-dependent PC-PLC and gamma-GT activities decreased significantly, together with alpha-fetoprotein expression. There was a close relationship between Ca2+-dependent PC-PLC and gamma-GT activities during differentiation as there was during proliferation. We suppose that Ca2+-dependent PC-PLC is involved in rat hepatocarcinogenesis induced by DEN and that it plays an important role in the phorbol ester-induced proliferation or retinoic acid-induced differentiation of liver cancer cells.

Animals↗