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Biomedical subjects

M Mao

Publications and source records attributed to M Mao.

At least 19 recordsLinked to original sources
PubMed2001

Tyrosine phosphorylation of p85 relieves its inhibitory activity on phosphatidylinositol 3-kinase.

Under resting conditions, the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K) serves to both stabilize and inactivate the p110 catalytic subunit. The inhibitory activity of p85 is relieved by occupancy of the NH(2)-terminal SH2 domain of p85 by phosphorylated tyrosine. Src family kinases phosphorylate tyrosine 688 in p85, a process that we have shown to be reversed by the activity of the p85-associated SH2 domain-containing phosphatase SHP1. We demonstrate that phosphorylation of the downstream PI3K target Akt is increased in cells lacking SHP1, implicating phosphorylation of p85 in the regulation of PI3K activity. Furthermore, the in vitro specific activity of PI3K associated with tyrosine- phosphorylated p85 is higher than that associated with nonphosphorylated p85. Expression of wild-type p85 inhibits PI3K enzyme activity as indicated by PI3K- dependent Akt phosphorylation. The inhibitory activity of p85 is accentuated by mutation of tyrosine 688 to alanine and reversed by mutation of tyrosine 688 to aspartic acid, changes that block and mimic tyrosine phosphorylation, respectively Strikingly, mutation of tyrosine 688 to aspartic acid completely reverses the inhibitory activity of p85 on cell viability and activation of the downstream targets Akt and NFkappaB, indicative of the physiological relevance of p85 phosphorylation. Tyrosine phosphorylation of Tyr(688) or mutation of tyrosine 688 to aspartic acid is sufficient to allow binding to the NH(2)-terminal SH2 domain of p85. Thus an intramolecular interaction between phosphorylated Tyr(688) and the NH(2)-terminal SH2 domain of p85 can relieve the inhibitory activity of p85 on p110. Taken together, the data indicate that phosphorylation of Tyr(688) in p85 leads to a novel mechanism of PI3K regulation.

Animals↗
PubMed2001

Experimental annotation of the human genome using microarray technology.

The most important product of the sequencing of a genome is a complete, accurate catalogue of genes and their products, primarily messenger RNA transcripts and their cognate proteins. Such a catalogue cannot be constructed by computational annotation alone; it requires experimental validation on a genome scale. Using 'exon' and 'tiling' arrays fabricated by ink-jet oligonucleotide synthesis, we devised an experimental approach to validate and refine computational gene predictions and define full-length transcripts on the basis of co-regulated expression of their exons. These methods can provide more accurate gene numbers and allow the detection of mRNA splice variants and identification of the tissue- and disease-specific conditions under which genes are expressed. We apply our technique to chromosome 22q under 69 experimental condition pairs, and to the entire human genome under two experimental conditions. We discuss implications for more comprehensive, consistent and reliable genome annotation, more efficient, full-length complementary DNA cloning strategies and application to complex diseases.

Algorithms↗
PubMed2001

Adenoviral delivery of osteoprotegerin ameliorates bone resorption in a mouse ovariectomy model of osteoporosis.

Osteoprotegerin (OPG) regulates bone resorption by inhibiting osteoclast formation, function, and survival. The current studies employed a mouse ovariectomy (OVX) model of estrogen deficiency to investigate gene therapy with OPG as a means of preventing osteoporosis. Young adult females injected with a recombinant adenoviral (Ad) vector carrying cDNA of either full-length OPG or a fusion protein combining the hOPG ligand-binding domain with the human immunoglobulin constant domain (Ad-hOPG-Fc) developed serum OPG concentrations exceeding the threshold needed for efficacy. However, elevated circulating OPG levels were sustained for up to 18 months only in mice given Ad-hOPG-Fc. Administration of Ad-hOPG-Fc titers between 10(7) and 10(9) pfu yielded dose-dependent increases in serum OPG. Mice subjected to OVX or sham surgery followed by immediate treatment with Ad-hOPG-Fc had significantly more bone volume with reduced osteoclast numbers in axial and appendicular bones after 4 weeks. In contrast, animals given OVX and either a control vector or vehicle had significantly less bone than did comparably treated sham-operated mice. This study demonstrates that a single adenoviral gene transfer can produce persistent high-level OPG expression and shows that gene therapy to provide sustained delivery of OPG may prove useful in treating osteoporosis.

Adenoviridae↗
PubMed2001

The role of genetic abnormalities of PTEN and the phosphatidylinositol 3-kinase pathway in breast and ovarian tumorigenesis, prognosis, and therapy.

Breast and ovarian cancers exhibit similar epidemiologic, genotypic, and phenotypic characteristics. Phosphatidylinositol 3-kinase (PI3K) and the PTEN tumor suppressor gene product phosphorylate and dephosphorylate the same 3' site in the inositol ring of membrane phosphatidylinositols. Germ-line mutations in the PTEN tumor suppressor gene are causative of Cowden's breast cancer predisposition syndrome, and PTEN is frequently mutated in sporadic breast cancers. In contrast, amplification of multiple components of the PI3K pathway is a hallmark of serous epithelial ovarian cancers. The resultant activation of the PI3K pathway in both breast and ovarian cancers contributes to cell-cycle progression, decreased apoptosis, and increased metastatic capabilities. Strikingly, both ovarian and breast cancer cells are selectively sensitive to pharmacologic and genetic manipulation of the PI3K pathway, making molecular therapeutics targeting this pathway particularly attractive approaches for these cancers.

Animals↗
PubMed2001

Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer.

We describe a flexible system for gene expression profiling using arrays of tens of thousands of oligonucleotides synthesized in situ by an ink-jet printing method employing standard phosphoramidite chemistry. We have characterized the dependence of hybridization specificity and sensitivity on parameters including oligonucleotide length, hybridization stringency, sequence identity, sample abundance, and sample preparation method. We find that 60-mer oligonucleotides reliably detect transcript ratios at one copy per cell in complex biological samples, and that ink-jet arrays are compatible with several different sample amplification and labeling techniques. Furthermore, results using only a single carefully selected oligonucleotide per gene correlate closely with those obtained using complementary DNA (cDNA) arrays. Most of the genes for which measurements differ are members of gene families that can only be distinguished by oligonucleotides. Because different oligonucleotide sequences can be specified for each array, we anticipate that ink-jet oligonucleotide array technology will be useful in a wide variety of DNA microarray applications.

Cells, Cultured↗
PubMed2001

[A study for optimal method of separating and cultivating embryonic rat cerebral neurons].

In order to improve the method of separating and cultivating the embryonic rat cerebral neurons in vitro, we compared different digestive enzymes, mechanical separating methods and two different mediums. It was found that highly purified and active embryonic rat cerebral neurons could be harvested by cultivating the neurons in the standard neurobasal medium with B-27 supplement, 10% horse serum, 10% fetal bovine serum and L-Glutamine after separated by digestion with the use of 0.05% trypsin and 0.05% collagenase II.

Animals↗
PubMed2001

[Level of nerve growth factor protein in cerebellar cortex of rat pups in intrauterine growth retardation model by clamping the uterine vasculature of pregnant rat].

OBJECTIVE: To understand the level of nerve growth factor (NGF) in the brain of rat pups in intrauterine growth retardation (IUGR), explore the mechanism of brain injury. METHODS: Animal models for IUGR pups were established by clamping the uterine vasculature of pregnant rats with 30 minutes in this study. NGF proteins were measured by immunohistochemistry in the cerebellar cortex of rat pups at birth (21 days of gestation), and on the sixth day after birth. RESULTS: 1. The body weight of the 21-day fetuses in IUGR group decreased 16.1% as compared to that in control group (P < 0.0001); 2. The brain weight of the 21-day fetuses in IUGR group decreased to that in control group (P < 0.05) development of their brain retardation during the intrauterine in IUGR fetus period in this model; 3. On the sixth day after birth, there was no statistical difference in body weight between the IUGR and the control groups. But the brain weight difference between these two groups was still there; 4. At birth and on the sixth day after birth, there were statistical differences in NGF proteins in cerebellum between the IUGR and the control groups, which got along with the decrease of their brain weights. CONCLUSION: A successful animal model of IUGR was established by clamping the uterine vasculature of pregnant rats with 30 minutes, and the development of their brain were retarded in IUGR fetus period in this model. Brain retardation in IUGR pups may relate with the decreased level of NGF in brain. This study discovered that quick "catch-up" growth in physical development and slow "catch-up" growth in brain development in IUGR pups.

Animals↗
PubMed2000

Inhibition of growth-factor-induced phosphorylation and activation of protein kinase B/Akt by atypical protein kinase C in breast cancer cells.

The protein kinase B/Akt serine/threonine kinase, located downstream of phosphoinositide 3-kinase (PI-3K), is a major regulator of cellular survival and proliferation. Atypical protein kinase C (aPKC) family members are activated by PI-3K and also contribute to cell proliferation, suggesting that Akt and aPKC might interact to activate signalling through the PI-3K cascade. Here we demonstrate that blocking PKC activity in MDA-MB-468 breast cancer cells increased the phosphorylation and activity of Akt. Functional PI-3K was required for the PKC inhibitors to increase Akt phosphorylation and activation, potentially owing to the activation of specific PKC isoforms by PI-3K. The concentration dependence of the action of the PKC inhibitors implicates aPKC in the inhibition of Akt phosphorylation and activity. In support of a role for aPKC in the regulation of Akt, Akt and PKCzeta or PKClambda/iota were readily co-precipitated from the BT-549 breast cancer cell line. Furthermore, the overexpression of PKCzeta inhibited growth-factor-induced increases in Akt phosphorylation and activity. Thus PKCzeta associates physically with Akt and decreases Akt phosphorylation and enzyme activity. The effects of PKC on Akt were transmitted through the PI-3K cascade as indicated by changes in p70 s6 kinase (p70(s6k)) phosphorylation. Thus PKCzeta, and potentially other PKC isoenzymes, regulate growth-factor-mediated Akt phosphorylation and activation, which is consistent with a generalized role for PKCzeta in limiting growth factor signalling through the PI-3K/Akt pathway.

Breast Neoplasms↗
PubMed2000

Gene expression networks underlying retinoic acid-induced differentiation of acute promyelocytic leukemia cells.

To elucidate the molecular mechanism of all-trans-retinoic acid (ATRA)-induced differentiation of acute promyelocytic leukemia (APL) cells, the gene expression patterns in the APL cell line NB(4) before and after ATRA treatment were analyzed using complementary DNA array, suppression-subtractive hybridization, and differential-display-polymerase chain reaction. A total of 169 genes, including 8 novel ones, were modulated by ATRA. The ATRA-induced gene expression profiles were in high accord with the differentiation and proliferation status of the NB(4) cells. The time courses of their modulation were interesting. Among the 100 up-regulated genes, the induction of expression occurred most frequently 12-48 hours after ATRA treatment, while 59 of 69 down-regulated genes found their expression suppressed within 8 hours. The transcriptional regulation of 8 induced and 24 repressed genes was not blocked by cycloheximide, which suggests that these genes may be direct targets of the ATRA signaling pathway. A balanced functional network seemed to emerge, and it formed the foundation of decreased cellular proliferation, maintenance of cell viability, increased protein modulation, and promotion of granulocytic maturation. Several cytosolic signaling pathways, including JAKs/STAT and MAPK, may also be implicated in the symphony of differentiation. (Blood. 2000;96:1496-1504)

Antineoplastic Agents↗
PubMed2000

Lysophospholipid growth factors in the initiation, progression, metastases, and management of ovarian cancer.

Levels of lysophosphatidic acid (LPA) and lysophosphatidylcholine (LPC) are elevated in the plasma and ascites of ovarian cancer patients, but not in most other tumor types. LPA increases cell proliferation, cell survival, resistance to cisplatin, cell shrinkage, and production of vascular endothelial growth factor, urokinase plasminogen activator, and LPA itself in ovarian cancer cells, but not in normal ovarian surface epithelial cells. PSP24 and members of the endothelial differentiation gene (EDG) family (EDG1, EDG2, EDG4, and EDG7) of G protein-coupled receptors mediate LPA signaling. Ovarian cancer cell lines do not express EDG1 mRNA, have variable EDG2 mRNA and protein levels, and frequently exhibit levels of EDG4 mRNA and protein, suggesting that EDG4 may contribute to the deleterious effects of LPA in ovarian cancer. In contrast, activation of the EDG2 LPA receptor on ovarian cancer cells may lead to apoptosis and counter the effects of other LPA receptors. Thus, the development of agonists and antagonists for the appropriate spectrum of LPA receptors may alter proliferation, apoptosis, or response to therapy of ovarian cancer cells. Indeed, over 60% of all current drugs target the G protein-coupled family of receptors, making the LPA receptor family a "drugable" target. LPC, although not as thoroughly studied, increases cellular proliferation and mediates multiple other functions through unique signaling pathways.

Ascites↗
PubMed2000

Constitutive and lysophosphatidic acid (LPA)-induced LPA production: role of phospholipase D and phospholipase A2.

Ascitic fluid and plasma from ovarian cancer patients, but not from patients with nongynecological tumors, contain elevated levels of the bioactive phospholipid lysophosphatidic acid (LPA). We show that ovarian cancer cells constitutively produce increased amounts of LPA as compared with normal ovarian epithelium, the precursor of ovarian epithelial cancer, or breast cancer cells. In addition, LPA, but not other growth factors, increases LPA production by the OVCAR-3 ovarian cancer cell line but not by normal ovarian epithelium or breast cancer cell lines. We show that phospholipase D activity contributes to both constitutive and LPA-induced LPA production by ovarian cancer cells. Constitutive and LPA-induced LPA synthesis by ovarian cancer cells is differentially regulated with respect to the requirement of specific phospholipase A2 (PLA2) subgroups. Group IB (pancreatic) secretory PLA2 plays a critical role in both constitutive and LPA-induced LPA formation, whereas group IIA (synovial) secretory PLA2 contributes to LPA-induced LPA production only. Calcium-dependent and/or -independent cytosolic PLA2s are required for constitutive LPA synthesis but do not play a role in LPA-induced LPA formation. LPA increases the proliferation of ovarian cancer cells, decreases sensitivity to cisplatin, the most commonly used drug in ovarian cancer, decreases apoptosis and anoikis, increases protease production, and increases production of neovascularization mediators. Thus, an understanding of the source and regulation of LPA production in ovarian cancer patients could identify novel targets for therapy.

Antineoplastic Agents↗
PubMed2000

[Exploration of gene expression profiles of CD(34)(+) hematopoietic stem/progenitor cells based on large scale sequencing].

OBJECTIVE: To set up a large scale sequencing system and explore the gene expression profiles of CD(34)(+) hematopoietic stem/progenitor cells (HSPCs). METHODS: CD(34)(+) cells were isolated from umbilical cord blood and subjected to cDNA library construction. A primary profile of gene expression in HSPCs was emerged by EST sequencing and bioinformatics analyzing. RESULTS: Among 9,866 ESTs thus obtained, 7 476 meaningful ESTs were clustered into 2,060 unique sequence species (USSs), representing 1,054 known gene species and 1,006 unknown gene fragments. The 1054 known genes were divided into 8 categories: (1) hematopoiesis associated: 73, (2) chromatin structure and cell division/apoptosis: 91, (3) signal transduction and receptors: 111, (4) cell structure/mobility: 48, (5) cell/organism defense/homeostasis: 41; (6) Gene expression (transcription, translation and modification): 265, (7) metabolism: 192; and (8) unclassified: 233. CONCLUSION: A gene expression profile including 1,054 known genes and 1006 new gene fragments of HSPCs was primarily obtained, which may lay a basis for the further study on the molecular mechanism of hematopoiesis regulation and provide candidates for new gene cloning.

Antigens, CD34↗
PubMed1999

Molecular cloning of six novel Krüppel-like zinc finger genes from hematopoietic cells and identification of a novel transregulatory domain KRNB.

To clone zinc finger genes expressed in hematopoietic system, we designed primers based on conserved Cys(2)/His(2) zinc finger sequences to amplify corresponding domains from mRNA of normal bone marrow and leukemia cell line NB4. DNA fragments of novel zinc finger genes were chosen and used as probe pool to screen cDNA libraries or subject to rapid amplification of cDNA ends in order to obtain full-length cDNA. Six cDNAs including whole open reading frame of zinc finger proteins, named as ZNF191, ZNF253 (BMZF-1), ZNF255 (BMZF-2), ZNF256 (BMZF-3), ZNF257 (BMZF-4), and ZNF254 (BMZF-5) were obtained. All six belong to the Krüppel-like zinc finger gene family, and typical transcriptional regulatory motifs exist in the N-terminal moiety, such as the SCAN box in ZNF191, and the KRAB domains in ZNF253, ZNF254, ZNF256, and ZNF257. A previously undefined sequence nominated as Krüppel-related novel box, which may represent a new transregulatory motif, was revealed at the N terminus of ZNF255. The transregulatory function of non-zinc finger regions of ZNF191, ZNF253, and ZNF255 were addressed in yeast and mammalian cells. The results indicated that ZNF255 might be a conditional transactivator, whereas ZNF253 and ZNF191 displayed a suppressive effect on the transcription in yeast and/or mammalian systems.

Amino Acid Sequence↗
PubMed1999

The PTEN/MMAC1/TEP tumor suppressor gene decreases cell growth and induces apoptosis and anoikis in breast cancer cells.

The PTEN/MMAC1/TEP (PTEN) tumor suppressor gene at 10q23.3 is mutated in multiple types of sporadic tumors including breast cancers and also in the germline of patients with the Cowden's breast cancer predisposition syndrome. The PTEN gene encodes a multifunctional phosphatase capable of dephosphorylating the same sites in membrane phosphatidylinositols phosphorylated by phosphatidylinositol 3'-kinase (PI3K). We demonstrate herein that loss of PTEN function in breast cancer cells results in an increase in basal levels of phosphorylation of multiple components of the P13K signaling cascade as well as an increase in duration of ligand-induced signaling through the P13K cascade. These alterations are reversed by wild-type but not phosphatase inactive PTEN. In the presence of high concentrations of serum, enforced expression of PTEN induces a predominant G1 arrest consistent with the capacity of PTEN to evoke increases in the expression of the p27Kip1 cyclin dependent kinase inhibitor. In the presence of low concentrations of serum, enforced PTEN expression results in a marked increase in cellular apoptosis, a finding which is consistent with the capacity of PTEN to alter the phosphorylation, and presumably function, of the AKT, BAD, p70S6 kinase and GSK3 alpha apoptosis regulators. Under anchorage-independent conditions, PTEN also induces anoikis, a form of apoptosis that occurs when cells are dissociated from the extracellular matrix, which is enhanced in conjunction with low serum culture conditions. Together, these data suggest that PTEN effects on the PI3K signaling cascade are influenced by the cell stimulatory context, and that depending on the exposure to growth factors and other exogenous stimuli such as integrin ligation, PTEN can induce cell cycle arrest, apoptosis or anoikis in breast cancer cells.

Apoptosis↗
PubMed1999

Regulation of BAD phosphorylation at serine 112 by the Ras-mitogen-activated protein kinase pathway.

The function of the pro-apoptotic molecule BAD is regulated by phosphorylation of two sites, serine-112 (Ser-112) and serine-136 (Ser-136). Phosphorylation at either site results in loss of the ability of BAD to heterodimerize with the survival proteins BCL-XL or BCL-2. Phosphorylated BAD binds to 14-3-3 and is sequestered in the cytoplasm. It has been shown that phosphorylation of BAD at Ser-136 is mediated by the serine/threonine protein kinase Akt-1/PKB which is downstream of phosphatidylinositol 3-kinase (PI3K). The signaling process leading to phophorylation of BAD at Ser-112 has not been identified. In this study, we show that phosphorylation of the two serine residues of BAD is differentially regulated. While Ser-136 phosphorylation is concordant with activation of Akt, Ser-112 phosphorylation does not correlate with Akt activation. Instead, we demonstrate that activated Ras and Raf, which are upstream of mitogen-activated protein kinases (MAPK), stimulate selective phosphorylation of BAD at Ser-112. Furthermore, phosphorylation of Ser-112, but not Ser-136 requires activation of the MAPK pathway as the MEK inhibitor, PD 98059, blocks EGF-, as well as activated Ras- or Raf-mediated phosphorylation of BAD at Ser-112. Therefore, the PI3K-Akt and Ras-MAPK pathways converge at BAD by mediating phosphorylation of distinct serine residues.

3T3 Cells↗
PubMed1999

SLUG, a ces-1-related zinc finger transcription factor gene with antiapoptotic activity, is a downstream target of the E2A-HLF oncoprotein.

The E2A-HLF fusion gene transforms human pro-B lymphocytes by interfering with an early step in apoptotic signaling. In a search for E2A-HLF-responsive genes, we identified a zinc finger transcription factor, SLUG, whose product belongs to the Snail family of developmental regulatory proteins. Importantly, SLUG bears close homology to the CES-1 protein of C. elegans, which acts downstream of CES-2 in a neuron-specific cell death pathway. Consistent with the postulated role of CES-1 as an antiapoptotic transcription factor, SLUG was nearly as active as Bcl-2 or Bcl-xL in promoting the survival of IL-3-dependent murine pro-B cells deprived of the cytokine. We conclude that SLUG is an evolutionarily conserved transcriptional repressor whose activation by E2A-HLF promotes the aberrant survival and eventual malignant transformation of mammalian pro-B cells otherwise slated for apoptotic death.

Animals↗
PubMed1999

Lysophosphatidic acid induces urokinase secretion by ovarian cancer cells.

Lysophosphatidic acid (LPA) is present at high concentrations in ascites from ovarian cancer patients and has potent mitogenic properties in vitro. Urokinase plasminogen activator (uPA), a critical component of the metastatic cascade, is also found at high concentrations in ovarian ascites and ovarian cancers, and the levels of uPA correlate inversely with prognosis. Because LPA stimulates the invasion of both hepatoma and lung cell lines, we investigated whether LPA could induce uPA secretion by ovarian epithelial cells and whether this process was associated with malignant transformation of ovarian epithelial cells. As indicated by zymography and Western blotting, physiologically relevant concentrations of LPA equivalent to those present in ovarian cancer ascites stimulated uPA secretion in the ovarian cancer cell lines OVCAR-3, SKOV-3, OVCA 429, OVCA 432, and OVCA 433, but not from established normal ovarian epithelial (NOE) cells as indicated by normal epithelial cell lines NOE 033 and NOE 035 or from SV40 large T antigen-immortalized normal epithelial cell lines IOSE 29 and IOSE 80. 18:1 LPA, but not 18:0 LPA, 16:0 LPA, or lysophosphatidylcholine, induced uPA secretion, concordant with previous studies of LPA receptor selectivity. Expression of the edg-2 LPA receptor was not consistently different between normal epithelial cell lines and ovarian cancer cell lines. In contrast, expression of the edg-4 LPA receptor was markedly increased in ovarian cancer cell lines as compared with NOE cell lines, raising the possibility that the edg-4 LPA receptor contributes to the ability of ovarian cancer cells but not NOE cells to produce uPA in response to LPA. LPA induced a consistent increase in uPA promoter activity and mRNA levels, suggesting that increased uPA production is, at least in part, transcriptional. Malignant transformation may alter LPA-induced cell activation by altering the pattern of LPA receptors present and may possibly lead to more aggressive behavior by up-regulating LPA-mediated uPA secretion and stimulating extracellular stromal breakdown and invasion.

Breast Neoplasms↗