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S Huang

Publications and source records attributed to S Huang.

At least 19 recordsLinked to original sources
PubMed2000

Induction of mitosis-mediated apoptosis by sodium arsenite in HeLa S3 cells.

Arsenic has been used effectively as a chemotherapeutic drug for the treatment of acute promyelocytic leukemia patients. Numerous studies have demonstrated that arsenic induces apoptosis in various cell types. In the present study, we showed that approximately 35% of arsenite-treated HeLa S3 cells arrested in mitosis. After release from arsenite treatment, more than 80% of arsenite-arrested mitotic cells subsequently underwent apoptosis, as indicated by anachronistic nuclear envelope reformation, DNA ladder occurrence, chromatin condensation, and activation of caspases 3 and 9. In exploring how these cells entered apoptosis mechanistically, we found an inverse correlation between mitotic indexes and apoptotic frequencies. As shown by using Percoll density gradient fractionation and flow cytometric analysis, the mitosis-mediated apoptosis induced by arsenite was accompanied by delayed cyclin B degradation and altered mitotic exit. Furthermore, treatment of arsenite-arrested mitotic cells with staurosporine or 2-aminopurine resulted in a rapid degradation of cyclin B, moved these cells forward to interphase without cell division, and abrogated apoptosis. These results suggest that apoptosis occurs in arsenite-arrested mitotic cells that exit mitosis abnormally.

Apoptosis↗
PubMed2000

Characterization of the human and mouse WRN 3'-->5' exonuclease.

Werner's syndrome (WS) is an autosomal recessive disorder in humans characterized by the premature development of a partial array of age-associated pathologies. WRN, the gene defective in WS, encodes a 1432 amino acid protein (hWRN) with intrinsic 3'-->5' DNA helicase activity. We recently showed that hWRN is also a 3'-->5' exonuclease. Here, we further characterize the hWRN exonuclease. hWRN efficiently degraded the 3' recessed strands of double-stranded DNA or a DNA-RNA heteroduplex. It had little or no activity on blunt-ended DNA, DNA with a 3' protruding strand, or single-stranded DNA. The hWRN exonuclease efficiently removed a mismatched nucleotide at a 3' recessed terminus, and was capable of initiating DNA degradation from a 12-nt gap, or a nick. We further show that the mouse WRN (mWRN) is also a 3'-->5' exonuclease, with substrate specificity similar to that of hWRN. Finally, we show that hWRN forms a trimer and interacts with the proliferating cell nuclear antigen in vitro. These findings provide new data on the biochemical activities of WRN that may help elucidate its role(s) in DNA metabolism.

Amino Acid Sequence↗
PubMed2000

Inhibition of tumor growth and metastasis of human melanoma by intracellular anti-ATF-1 single chain Fv fragment.

Activating transcription factor-1 (ATF-1) and cAMP-responsive element (CRE)-binding protein (CREB) have been implicated in cAMP and Ca2+-induced transcriptional activation. The expression of the transcription factors CREB and ATF-1 is upregulated in metastatic melanoma cells. However, how overexpression of ATF-1/CREB contributes to the acquisition of the metastatic phenotype remains unclear. Here, the effect of disrupting ATF-1 activity was investigated using intracellular expression of an inhibitory anti-ATF-1 single chain antibody fragment (ScFv). Intracellular expression of ScFv anti-ATF-1 in MeWo melanoma cells caused significant reduction in CRE-dependent promoter activation. In addition, expression of ScFv anti-ATF-1 in melanoma cells suppressed their tumorigenicity and metastatic potential in nude mice. ScFv anti-ATF-1 rendered the melanoma cells susceptible to thapsigargin-induced apoptosis in vitro and caused massive apoptosis in tumors transplanted subcutaneously into nude mice, suggesting that ATF-1 and its associated proteins act as survival factor for human melanoma cells. This is the first report to demonstrate the potential of ScFv anti-ATF-1 as an inhibitor of tumor growth and metastasis of solid tumor in vivo. Oncogene (2000).

Activating Transcription Factor 1↗
PubMed2000

Intrinsic bias and lineage restriction in the phenotype determination of dopamine and neuropeptide Y amacrine cells.

Blastomere lineages are differentially biased to produce different neurotransmitter subtypes of amacrine cells (Huang and Moody, 1995, 1997,). To elucidate when this bias is acquired, we examined amacrine lineages at different early developmental times. Our experiments demonstrate that the bias to express dopamine and neuropeptide Y amacrine fates involves several steps before the formation of the definitive optic cup. At cleavage stages, a retinal progenitor that contributes large numbers of cells is already biased to produce its normal repertoire of dopamine amacrine cells, as revealed by transplantation to a new location, whereas the amacrine fate of a progenitor that contributes fewer cells is modified by its new position. At neural plate stages, not all retinal progenitors are multipotent. Nearly one-half populate only the inner nuclear layer and are enriched in amacrine cells. During early optic vesicle stages, an appropriate mitotic tree is required for dopamine and neuropeptide Y, but not serotonin, amacrine cell clusters to form. Thus, the acquisition of amacrine fate bias involves intrinsic maternal factors at cleavage, fate restriction in the neural plate, and specified mitotic patterns in the optic vesicle. At each of these steps only a subset of the embryonic retinal progenitors contributing to amacrine subtypes is biased; the remaining progenitors maintain multipotency. Thus, from the earliest embryonic stages, progenitors of the retina are a dynamic mosaic. This is the first experimental demonstration of amacrine fate decisions that occur during early embryonic periods in advance of the events described in the later, committed retina.

Animals↗
PubMed2000

Urokinase plasminogen activator/urokinase-specific surface receptor expression and matrix invasion by breast cancer cells requires constitutive p38alpha mitogen-activated protein kinase activity.

Overexpression of urokinase plasminogen activator (uPA) and its receptor (uPAR) has been well documented in a wide variety of tumor cells. In breast cancer, expression of uPA/uPAR is essential for tumor cell invasion and metastasis. However, the mechanism responsible for uPA/uPAR expression in cancer cells remains unclear. In the studies reported here, we show that endogenous p38 MAPK activity correlates well with breast carcinoma cell invasiveness. Treatment of highly invasive BT549 cells with a specific p38 MAPK inhibitor SB203580 diminished both uPA/uPAR mRNA and protein expression and abrogated the ability of these cells to invade matrigel, suggesting that p38 MAPK signaling pathway is involved in the regulation of uPA/uPAR expression and breast cancer cell invasion. We also demonstrated that SB203580-induced reduction in uPA/uPAR mRNA expression resulted from the de- stabilization of uPA and uPAR mRNA. Finally, by selectively inhibiting p38alpha or p38beta MAPK isoforms, we demonstrate that p38alpha, rather than p38beta, MAPK activity is essential for uPA/uPAR expression. These studies suggest that p38alpha MAPK signaling pathway is important for the maintenance of breast cancer invasive phenotype by promoting the stabilities of uPA and uPAR mRNA.

Breast Neoplasms↗
PubMed2000

Cell growth and matrix invasion of EBV-immortalized human B lymphocytes is regulated by expression of alpha(v) integrins.

alpha(v) integrins have been shown to play an important role in epithelial-derived cell migration, cell growth and tumor invasion/metastasis, however their role on cells of hematopoietic origin is less clear. Epstein-Barr virus (EBV), a human herpesvirus associated with several lymphoproliferative disorders in man, induces expression of alpha(v) integrins on transformed B lymphocytes. In the studies reported here, we show that EBV infection increases alpha(v), beta3 and beta5 integrin subunit mRNAs as well as upregulates the expression of the alphavbeta3 integrin protein on human B cells. Among the nine different EBV proteins expressed in latently infected B cells (nuclear and plasma membrane-associated), only LMP1, LMP2A and EBNA2 were shown to selectively transactivate the alpha(v) integrin promoter. Treatment of EBV-transformed B cells with alpha(v) antisense oligonucleotides specifically reduced cell surface expression of alpha(v) integrins, inhibited cell growth in low serum, reduced cell invasion in matrigels and decreased expression of metalloprotease, MMP9. These studies indicate that alpha(v) integrins play a significant role in EBV-induced B-lymphocyte proliferation and invasion. Strategies to interfere with alphav integrin expression and/or function may therefore be of potential value in the treatment of EBV-associated lymphoproliferative disorders.

Antigens, CD↗
PubMed2000

Patterned deposition of cells and proteins onto surfaces by using three-dimensional microfluidic systems.

Three-dimensional microfluidic systems were fabricated and used to pattern proteins and mammalian cells on a planar substrate. The three-dimensional topology of the microfluidic network in the stamp makes this technique a versatile one with which to pattern multiple types of proteins and cells in complex, discontinuous structures on a surface. The channel structure, formed by the stamp when it is in contact with the surface of the substrate, limits migration and growth of cells in the channels. With the channel structure in contact with the surface, the cells stop dividing once they form a confluent layer. Removal of the stamp permits the cells to spread and divide.

Animals↗
PubMed2000

Candidate tumor suppressor RIZ is frequently involved in colorectal carcinogenesis.

The distal portion of chromosome 1p is one of the most commonly affected regions in human cancer. In this study of hereditary and sporadic colorectal cancer, a region of frequent deletion was identified at 32.2 centimorgans from 1ptel. Deletion breakpoints clustered in the vicinity of or inside the gene RIZ, which encodes a retinoblastoma protein-interacting zinc finger protein. Sequence analysis revealed frequent frameshift mutations of the RIZ gene. The mutations consisted of 1- or 2-bp deletions of a coding (A)(8) or (A)(9) tract and were confined to microsatellite-unstable colorectal tumors, being present in 9 of 24 (37.5%) primary tumors and in 6 of 11 (54.5%) cell lines; in 2 cell lines the mutation was homozygous/hemizygous. The mutations apparently were selected clonally in tumorigenesis, because similar poly(A) tracts in other genes were not affected. Two alternative products of the gene exist, RIZ1, which contains a PR (PRDI-BF1-RIZ1) domain implicated in tumor suppressor function, and RIZ2, which is lacking this motif. Furthermore, the C-terminal region, which contains the poly(A) tracts, includes a PR-binding motif, possibly mediating interactions with other proteins or with RIZ itself (oligomerization). Four of eleven microsatellite-unstable colorectal cancer cell lines, three of which had frameshifts, showed reduced or absent mRNA expression of RIZ1. In a cell line that is homozygous/hemizygous for the typical frameshift mutation, immunoblotting showed truncated RIZ protein, whereas adenovirus-mediated RIZ1 expression caused G(2)/M arrest and apoptosis. We propose that RIZ is a target of the observed 1p alterations, with impairment of the PR domain-mediated function through either frameshift mutation or genomic deletion.

Apoptosis↗
PubMed2000

The LIM homeobox gene Lhx9 is essential for mouse gonad formation.

During mammalian embryonic development, the ovaries and testes develop from somatic cells of the urogenital ridges as indifferent gonads, harbouring primordial germ cells that have migrated there. After sex determination of the gonads, the testes produce testosterone and anti-Mullerian hormone which mediate male sexual differentiation, and the female developmental pathway ensues in their absence. Here we show that transcripts of the LIM homeobox gene Lhx9 are present in urogenital ridges of mice at embryonic day 9.5; later they localize to the interstitial region as morphological differentiation occurs. In mice lacking Lhx9 function, germ cells migrate normally, but somatic cells of the genital ridge fail to proliferate and a discrete gonad fails to form. In the absence of testosterone and anti-Mullerian hormone, genetically male mice are phenotypically female. The expression of steroidogenic factor 1 (Sf1), a nuclear receptor essential for gonadogenesis, is reduced to minimal levels in the Lhx9-deficient genital ridge, indicating that Lhx9 may lie upstream of Sf1 in a developmental cascade. Unlike mice lacking other genes that mediate early stages of gonadogenesis, Lhx9 mutants do not exhibit additional major developmental defects. Thus, LHX9 mutations may underlie certain forms of isolated gonadal agenesis in humans.

Animals↗
PubMed2000

Differential regulation of the phosphatidylinositol 3-kinase/Akt and p70 S6 kinase pathways by the alpha(1A)-adrenergic receptor in rat-1 fibroblasts.

Phosphatidylinositol (PI) 3-kinase and its downstream effector Akt are thought to be signaling intermediates that link cell surface receptors to p70 S6 kinase. We examined the effect of a G(q)-coupled receptor on PI 3-kinase/Akt signaling and p70 S6 kinase activation using Rat-1 fibroblasts stably expressing the human alpha(1A)-adrenergic receptor. Treatment of the cells with phenylephrine, a specific alpha(1)-adrenergic receptor agonist, activated p70 S6 kinase but did not activate PI 3-kinase or any of the three known isoforms of Akt. Furthermore, phenylephrine blocked the insulin-like growth factor-I (IGF-I)-induced activation of PI 3-kinase and the phosphorylation and activation of Akt-1. The effect of phenylephrine was not confined to signaling pathways that include insulin receptor substrate-1, as the alpha(1)-adrenergic receptor agonist also inhibited the platelet-derived growth factor-induced activation of PI 3-kinase and Akt-1. Although increasing the intracellular Ca(2+) concentration with the ionophore A23187 inhibited the activation of Akt-1 by IGF-I, Ca(2+) does not appear to play a role in the phenylephrine-mediated inhibition of the PI 3-kinase/Akt pathway. The differential ability of phenylephrine and IGF-I to activate Akt-1 resulted in a differential ability to protect cells from UV-induced apoptosis. These results demonstrate that activation of p70 S6 kinase by the alpha(1A)-adrenergic receptor in Rat-1 fibroblasts occurs in the absence of PI 3-kinase/Akt signaling. Furthermore, this receptor negatively regulates the PI 3-kinase/Akt pathway, resulting in enhanced cell death following apoptotic insult.

Animals↗
PubMed2000

Temperature trends over the past five centuries reconstructed from borehole temperatures

For an accurate assessment of the relative roles of natural variability and anthropogenic influence in the Earth's climate, reconstructions of past temperatures from the pre-industrial as well as the industrial period are essential. But instrumental records are typically available for no more than the past 150 years. Therefore reconstructions of pre-industrial climate rely principally on traditional climate proxy records, each with particular strengths and limitations in representing climatic variability. Subsurface temperatures comprise an independent archive of past surface temperature changes that is complementary to both the instrumental record and the climate proxies. Here we use present-day temperatures in 616 boreholes from all continents except Antarctica to reconstruct century-long trends in temperatures over the past 500 years at global, hemispheric and continental scales. The results confirm the unusual warming of the twentieth century revealed by the instrumental record, but suggest that the cumulative change over the past five centuries amounts to about 1 K, exceeding recent estimates from conventional climate proxies. The strength of temperature reconstructions from boreholes lies in the detection of long-term trends, complementary to conventional climate proxies, but to obtain a complete picture of past warming, the differences between the approaches need to be investigated in detail.

Journal Article↗
PubMed2000

The Smad4 activation domain (SAD) is a proline-rich, p300-dependent transcriptional activation domain.

Transforming growth factor-beta (TGF-beta) family members signal through a unique set of intracellular proteins called Smads. Smad4, previously identified as the tumor suppressor DPC4, is functionally distinct among the Smad family, and is required for the assembly and transcriptional activation of diverse, Smad-DNA complexes. We previously identified a 48-amino acid proline-rich regulatory element within the middle linker domain of this molecule, the Smad4 activation domain (SAD), which is essential for mediating these signaling activities. We now characterize the functional activity of the SAD. Mutants lacking the SAD are still able to form complexes with other Smad family members and associated transcription factors, but cannot activate transcription in these complexes. Furthermore, the SAD itself is able to activate transcription in heterologous reporter assays, identifying it as a proline-rich transcriptional activation domain, and indicating that the SAD is both necessary and sufficient to activate Smad-dependent transcriptional responses. We show that transcriptional activation by the SAD is p300-dependent, and demonstrate that this activity is associated with a physical interaction of the SAD with the amino terminus of p300. These data identify a novel function of the middle linker region of Smad4, and define the role of the SAD as an important locus determining the transcriptional activation of the Smad complex.

Amino Acid Sequence↗
PubMed2000

Review: perinucleolar structures.

Several perinucleolar structures have been described in recent years. This review primarily summarizes recent studies regarding two of these structures, the perinucleolar compartment (PNC) and the Sam68 nuclear body (SNB). A number of studies have explored their ultrastructure and molecular components. Despite their different nuclear localizations, PNCs and SNBs share some common characteristics. They both are enriched with RNA binding proteins and nucleic acids and are predominantly localized to the periphery of the nucleolus. They are observed mostly in transformed cells, although prevalence differs among different cell types and cell lines. Their structural integrity is influenced by the transcriptional state of the cell. However, the functions of both the PNC and the SNB remain unknown. In addition to the PNC and SNB, a perinucleolar structure immunolabeled with an antibody to hnRNP L will be discussed.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Urinary NMP22 and renal cell carcinoma.

OBJECTIVES: To demonstrate the incidence of positive urinary nuclear matrix protein 22 (NMP22) values associated with renal cell carcinoma (RCC) of the kidney. Currently, urinary NMP22 is used to detect recurrent transitional cell carcinoma of the bladder. METHODS: From May 1997 to March 1998, urinary NMP22 values were obtained from 65 patients who had undergone either computed tomography scanning of the abdomen or renal ultrasound. Of the 65, 32 presented with solid renal masses. These patients underwent radical or partial nephrectomy; subsequent pathologic examination revealed that 30 had RCC. Two patients had oncocytomas. The remaining 33 patients presented with blunt abdominal trauma or abdominal pain or for follow-up for a urologic problem (a kidney stone or benign renal cyst). These patients had no evidence of renal malignancy on imaging and were used as controls. RESULTS: The urinary NMP22 values of patients with RCC were significantly higher than the values found in the control group (13.69 +/- 8.40 U/mL versus 3.03 +/- 2.70 U/mL, P <0.0078). Of the 30 patients with RCC, 12 (40%) had positive urinary NMP22 values of 10 U/mL or above. Chi-square analysis revealed a significant difference between the NMP22 values of the two groups (P <0.005). There were two false-positive NMP22 values in the 35 control patients. CONCLUSIONS: Urinary NMP22 values used to detect transitional cell carcinoma in individuals with previously diagnosed bladder cancer should be more broadly evaluated, since elevations have also been found to occur in RCC of the kidney. This finding may result in an increase in the incidental discovery of RCC. Future, more specific, NMP assays may hold promise for the detection of RCC.

Biomarkers, Tumor↗
PubMed2000

Synthesis, characterization and cytotoxicity of new platinum(IV) axial carboxylate complexes: crystal structure of potential antitumor agent [PtIV(trans-1R,2R-diaminocyclohexane)trans(acetate)2Cl2].

A series of new platinum(IV) complexes of the type [PtIV(DACH)trans(L)2Cl2] (where DACH = trans-1R,2R-diaminocyclohexane, and L = acetate, propionate, butyrate, valerate, hexanoate, or heptanoate) bearing the carboxylate groups in the axial positions have been synthesized and characterized by elemental analysis, IR, and 195Pt NMR spectroscopy. The crystal structure of the analogue [PtIV(DACH)trans(acetate)2Cl2] was determined by single crystal X-ray diffraction method. There were two crystallographically independent molecules, both of which lie on crystallographic two-fold axes. The bond lengths and bond angles of both the molecules were the same within the experimental error. The compound crystallizes in the monoclinic space group C2, with a = 11.180(2) A, b = 14.736(3) A, c = 10.644(2) A, beta = 112.38(3) degrees, Z = 4 and R = 0.0336, based upon a total of 1648 collected reflections. In this complex, the platinum had a slightly distorted octahedron geometry owing to the presence of a geometrically strained five-member ring. The two adjacent corners of the platinum plane were occupied by the two amino nitrogens of DACH, whereas the other two equatorial positions were occupied by two chloride ions. The remaining two axial positions were occupied by the oxygens of acetate ligands. The DACH ring was in a chair configuration. An intricate network of intermolecular hydrogen bonds held the crystal lattice together. These analogues were evaluated in vitro and demonstrated cytotoxic activity against the human ovarian 2008 tumor cell line (IC50 = 0.001-0.06 microM). Structure-activity study revealed that activity was highest for the analogue where L = butyrate.

Antineoplastic Agents↗
PubMed2000

Level of interleukin-8 expression by metastatic human melanoma cells directly correlates with constitutive NF-kappaB activity.

The purpose of this study was to determine whether constitutive NF-kappaB activity regulates the expression level of interleukin-8 (IL-8) in metastatic human melanoma cells. Cultures of metastatic human A375 melanoma cells expressed higher levels of IL-8 mRNA and protein than nonmetastatic A375 human melanoma cells. No discernible differences in IL-8 half-life were found between metastatic and nonmetastatic cells, but cells that overexpressed IL-8 had a higher transcription rate and increased IL-8 promoter activity. Analysis of the IL-8 promoter using deletion mutants revealed that the region within -133 was essential for constitutive IL-8 promoter activity and that mutation of NF-kappaB binding sites eliminated the constitutive IL-8 promoter activity. The activation of constitutive IL-8 transcription directly correlated with the level of constitutive NF-kappaB activity. Transfection of melanoma cells with a dominant-negative mutant IkappaBalpha expression vector (pLXSN-IkappaBalphaM) significantly decreased the level of constitutive NF-kappaB activity and expression of IL-8, demonstrating that constitutive NF-kappaB/relA activities contribute to overexpression of IL-8 in highly metastatic human melanoma cells.

Base Sequence↗
PubMed2000

Functional coupling of Ca(2+) channels to ryanodine receptors at presynaptic terminals. Amplification of exocytosis and plasticity.

Ca(2+)-induced Ca(2+) release (CICR) enhances a variety of cellular Ca(2+) signaling and functions. How CICR affects impulse-evoked transmitter release is unknown. At frog motor nerve terminals, repetitive Ca(2+) entries slowly prime and subsequently activate the mechanism of CICR via ryanodine receptors and asynchronous exocytosis of transmitters. Further Ca(2+) entry inactivates the CICR mechanism and the absence of Ca(2+) entry for >1 min results in its slow depriming. We now report here that the activation of this unique CICR markedly enhances impulse-evoked exocytosis of transmitter. The conditioning nerve stimulation (10-20 Hz, 2-10 min) that primes the CICR mechanism produced the marked enhancement of the amplitude and quantal content of end-plate potentials (EPPs) that decayed double exponentially with time constants of 1.85 and 10 min. The enhancement was blocked by inhibitors of ryanodine receptors and was accompanied by a slight prolongation of the peak times of EPP and the end-plate currents estimated from deconvolution of EPP. The conditioning nerve stimulation also enhanced single impulse- and tetanus-induced rises in intracellular Ca(2+) in the terminals with little change in time course. There was no change in the rate of growth of the amplitudes of EPPs in a short train after the conditioning stimulation. On the other hand, the augmentation and potentiation of EPP were enhanced, and then decreased in parallel with changes in intraterminal Ca(2+) during repetition of tetani. The results suggest that ryanodine receptors exist close to voltage-gated Ca(2+) channels in the presynaptic terminals and amplify the impulse-evoked exocytosis and its plasticity via CICR after Ca(2+)-dependent priming.

Animals↗