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Biomedical subjects

W W Li

Publications and source records attributed to W W Li.

At least 37 records · Page 2Linked to original sources
PubMed1999

The clinical manipulation of angiogenesis: pathology, side-effects, surprises, and opportunities with novel human therapies.

The first phase of angiogenesis research has provided knowledge of the basic pathobiology of angiogenesis and its manipulation in models, mouse, and man. The first line of therapeutic substances has been devised and is now in clinical trials. New lessons are being learned from clinical observations. Unexpected side-effects are being noted, particularly affecting the nervous system. Other side-effects may be anticipated from a sound knowledge of clinical pathology and recognition of the commonality of angiogenesis to multiple disease mechanisms, but these may be tolerable or avoidable. Angiogenesis researchers await further feedback and ideas from the clinic to stimulate the next phase of basic and applied research.

Angiogenesis Inhibitors↗
PubMed1999

Mechanisms of methotrexate resistance in acute leukemia. Decreased transport and polyglutamylation.

Drug resistance limits the effectiveness of methotrexate (MTX) for the treatment of acute leukemia. An increased understanding of the pathways involved in folate metabolism has allowed investigations of the mechanisms of resistance observed in leukemic blasts obtained from patients. Acute lymphocytic leukemia (ALL) was studied for mechanisms of acquired MTX resistance. MTX transport in 27 patients with untreated ALL and 31 patients with relapsed ALL was measured using a previously described competitive displacement assay. Only 13% of the untreated patients were considered to have impaired MTX transport whereas over 70% of the relapsed patients had evidence of impaired MTX transport. Northern analyses and quantitative RT-PCR for the reduced folate carrier (RFC) were performed on the RNA available from the leukemic blasts of 24 patients in whom MTX transport had been measured. Six of 9 samples with impaired MTX transport had decreased RFC expression (one had no detectable RFC expression), while three had no decrease in RFC expression. Acute myelocytic leukemia (AML) was studied to determine the basis of the decreased MTX polyglutamylation. Enzyme kinetics of the enzyme folylpolyglutamate synthetase (FPGS) were studied, demonstrating FPGS in the myeloid cell lines and patient samples had a higher K(m) for MTX as a substrate than lymphoid cells. Measuring gamma-glutamyl hydrolase enzyme activity allowed a more accurate prediction of steady state levels of MTX polyglutamates. A knowledge of the mechanisms of MTX resistance that occur in leukemic blasts obtained from patients may allow the development of therapeutic strategies to circumvent resistance.

Antimetabolites, Antineoplastic↗
PubMed1998

Off-label dermatologic therapies. Usage, risks, and mechanisms.

Off-label refers to the prescribing of Food and Drug Administration-approved drugs for a use not indicated on the package insert. The prescribing of off-label drugs may benefit patients with many dermatologic diseases including angiogenesis-related conditions. We surveyed 55 dermatologists from a single large academic program to assess their use of particular drugs for specific skin conditions, their perception of such use as being for Food and Drug Administration-approved or for off-label indications, and their attitudes towards off-label therapies. The practice of prescribing off-label drugs was common among the respondents, many of whom had misperceptions about which conditions are Food and Drug Administration-approved indications and about the legal ramifications of off-label therapies. We suggest that understanding the principles of off-label prescribing in conjunction with the mechanisms of drug action in diseases may help clinicians exercise their judgment in finding innovative therapies for their patients.

Dermatologic Agents↗
PubMed1997

Overexpression of p21waf1 leads to increased inhibition of E2F-1 phosphorylation and sensitivity to anticancer drugs in retinoblastoma-negative human sarcoma cells.

The effect of overexpression of p21waf1 on drug sensitivity was studied in an osteosarcoma cell line (SaOs-2) lacking both p53 and functional retinoblastoma protein using a tetracycline (TC)-inducible expression system. p21waf1 expression was barely detectable in SaOS-2 cells incubated in the presence of TC. After TC withdrawal, high levels of p21waf1 were induced in these cells. These p21waf1-induced cells showed increased sensitivity to doxorubicin, tomudex, and methotrexate as compared to uninduced cells; this condition is associated with increased apoptosis. Expression of p21waf1 reduced cyclin A-associated kinase activity and, surprisingly, resulted in inhibition of phosphorylation of E2F-1 and increased E2F-1 binding activity. An S-G2 cell cycle arrest/delay and an increase in expression of E2F-responsive genes (dihydrofolate reductase and thymidylate synthase) was correspondingly observed. Overexpression of p21waf1 in cells lacking functional retinoblastoma protein may mediate sensitivity to anticancer drugs by inhibiting E2F-1 phosphorylation, which may contribute to increased S-G2 cell cycle delay and increased cell susceptibility to apoptosis.

Anti-Bacterial Agents↗
PubMed1997

Capillary electrophoresis of methotrexate polyglutamates and its application in evaluation of gamma-glutamyl hydrolase activity.

A rapid and sensitive procedure for the separation of methotrexate (MTX) polyglutamates2 using capillary electrophoresis (CE) is described as it applies to the in vitro assay of the enzyme gamma-glutamyl hydrolase (GGH, EC 3.4.22.12). Distinct separation of MTX and polyglutamylated forms (up to glu4) is achieved within 10 min using a 75 microns I.D. capillary (50 cm, +25 kV), and enables quantitation of both reactant and enzyme products. As activity can be reliably determined using less than 5 x 10(5) eukaryotic cells, this new technique can be used to measure GGH in patient tumor samples and investigate the relationship between GGH levels and clinical MTX resistance.

Electrophoresis, Capillary↗
PubMed1997

Factors controlling ocular angiogenesis.

PURPOSE: To provide an overview of the cellular and molecular factors involved in ocular angiogenesis. METHODS: A literature search and review encompassing a broad range of medical and basic science disciplines was undertaken to survey contemporary insights into the mechanisms of angiogenic stimulation and inhibition. RESULTS: Ocular angiogenesis is a complex pathophysiologic process. Factors have been isolated that play key roles in the regulation of angiogenesis. The influence of stimulating growth factors is counterbalanced by a number of antiproliferative agents. The net result of these opposing factors on the vascular endothelial cell determines the outcome of angiogenesis homeostasis. Both endogenous and synthetic molecules can regulate ocular angiogenesis. CONCLUSIONS: The isolation and synthesis of molecular regulators of angiogenesis has enhanced our understanding of this process. Clinical trials are underway to determine the efficacy of these agents in controlling pathologic angiogenesis, including that in ocular disease.

Endothelium, Vascular↗
PubMed1997

gamma-Glutamyl hydrolase from human sarcoma HT-1080 cells: characterization and inhibition by glutamine antagonists.

Elevated gamma-glutamyl hydrolase (GGH) activity as a contributing factor in mechanisms of acquired and intrinsic antifolate resistance has been reported for several cultured cell lines. Despite this, little is known about this enzyme, especially the human species. Using the human HT-1080 sarcoma line, we observed the secretion of GGH activity into media during culture (a phenomenon that could be markedly stimulated by exposure to NH4Cl) and an acidic pH optimum for in vitro catalytic activity of the enzyme. These properties are consistent with a lysosomal location for the enzyme. Unlike rodent GGH, preparations of HT-1080 enzyme (purified < or = 2000-fold) displayed exopeptidase activity in cleaving successive end-terminal gamma-glutamyl groups from poly-L-gamma-glutamyl derivatives of folate, methotrexate (MTX), and para-aminobenzoic acid substrates and a marked preference for long-chain polyglutamates (Km values for glu4 versus glu1 derivatives were 17- and 15-fold lower for folate and MTX versions, respectively). Using an in vitro assay screen, several glutamine antagonists [i.e., 6-diazo-5-oxo-norleucine (DON), acivicin, and azaserine] were identified as human GGH inhibitors, with DON being the most potent and displaying time-dependent inhibition. In cell culture experiments, simultaneous exposure of DON (10 microM) and [3H]MTX for 24 hr resulted in modest elevations of the long-chain gamma-glutamyl derivatives of the antifolate for HT-1080 and another human sarcoma line. These compounds may serve as useful lead compounds in the development of specific GGH inhibitors for use in examining the relationship between GGH activity and antifolate action and may potentially be used in clinical combination with antifolates that require polyglutamylation for effective cellular retention.

Azaserine↗
PubMed1997

Induction of the mammalian GRP78/BiP gene by Ca2+ depletion and formation of aberrant proteins: activation of the conserved stress-inducible grp core promoter element by the human nuclear factor YY1.

Previously, we have identified a constitutive nuclear factor, p70CORE, from HeLa cell nuclear extract which interacts specifically with the stress-inducible change region (SICR) of the grp78 promoter. Here we report that p70CORE is identical to YY1, a member of the GLI zinc finger family, by criteria of biochemical properties including apparent molecular weight, binding site homology, immunoreactivity, and affinity purification. Recombinant YY1 binds the double-stranded SICR with high specificity but has no affinity for its single-stranded form. In cotransfection studies, YY1 specifically enhanced the transcriptional activation of the grp78 promoter under a variety of stress conditions: depletion of the endoplasmic reticulum calcium stores, protein glycosylation block, and formation of aberrant proteins by azetidine treatment. In contrast, YY1 has minimal effect on the stress induction of the hsp70 promoter. YY1 enhancement of the grp78 stress response is dependent on its DNA-binding domain, with little effect on the basal expression of the promoter. The effect of YY1 transactivation may be mediated by the highly conserved grp78 core element. This is the first example of the ubiquitous factor YY1 involved in regulating inducible gene expression and its involvement in mediating stress signals generated from the endoplasmic reticulum to the nucleus.

Azetidines↗
PubMed1997

Suppression of grp78 core promoter element-mediated stress induction by the dbpA and dbpB (YB-1) cold shock domain proteins.

The highly conserved grp78 core promoter element plays an important role in the induction of grp78 under diverse stress signals. Previous studies have established a functional region in the 3' half of the core (stress-inducible change region [SICR]) which exhibits stress-inducible changes in stressed nuclei. The human transcription factor YY1 is shown to bind the SICR and transactivate the core element under stress conditions. Here we report that expression library screening with the core element has identified two new core binding proteins, YB-1 and dbpA. Both proteins belong to the Y-box family of proteins characterized by an evolutionarily conserved DNA binding motif, the cold shock domain (CSD). In contrast to YY1, which binds only double-stranded SICR, the Y-box/CSD proteins much prefer the lower strand of the SICR. The Y-box proteins can repress the inducibility of the grp78 core element mediated by treatment of cells with A23187, thapsigargin, and tunicamycin. In gel shift assays, YY1 binding to the core element is inhibited by either YB-1 or dbpA. A yeast interaction trap screen using LexA-YY1 as a bait and a HeLa cell cDNA-acid patch fusion library identified YB-1 as a YY1-interacting protein. In cotransfection experiments, the Y-box proteins antagonize the YY1-mediated enhancement of transcription directed by the grp78 core in stressed cells. Thus, the CSD proteins may be part of the stress signal transduction mechanism in the mammalian system.

Bacterial Proteins↗
PubMed1997

A pathologic study of degeneration of the rod and cone populations of the rhodopsin Pro347Leu transgenic pigs.

PURPOSE: Transgenic pigs with rhodopsin (Pro347Leu) mutation exhibited rod-cone degeneration. We compared the pathologic characteristics of the rod degeneration versus those of the cone cells. METHODS: The posterior and peripheral retinas of these transgenic pigs of age 4, 6, 8, 12, 24 and 33 weeks and normal pigs of age 4 and 8 weeks were studied by light and EM and morphometry. RESULTS: The pathologic changes observed in the posterior and peripheral retinas of the transgenic pigs could be conveniently described in 3 phases: I) an initial phase of rapid and extensive degeneration of the rod cells in the first 6 weeks of age; II) an acute phase of cone cell degeneration involving approximately half of the population and lingering rod degeneration in the 6 to 12 weeks of age; and III) a partial cone recovery to be followed by a chronic degenerative phase of the remaining cones cells from 12 to 33 weeks of age. CONCLUSION: Our study showed that the degenerative changes of rod cells could be differentiated from those of the cone cells. Cone and rod populations degenerated along different time schedule with different pathologic features. Hence, treatment for retinitis pigmentosa might vary with the different stages of the disease.

Animals↗
PubMed1996

Establishment, characterization and drug sensitivity of four new human soft tissue sarcoma cell lines.

Four new cell lines were established from patients with soft tissue sarcomas. Drug sensitivity as well as genotypic characterization, which may be related to drug sensitivity in these cell lines, was determined. Karyotype, H-ras, c-myc and mutant p53 gene expression, Rb, G1- and S-phase cyclins, E2F and major cyclin/CDK inhibitors such as p16 and p21 and p-glycoprotein were analyzed using cytogenetic, Northern blot and immunological methods. Drug sensitivity was determined using growth inhibition tests. These cell lines differed in their morphology and growth rates, forming colonies in soft agar with a cloning efficiency of 4.3-13.4%, and 3 of the 4 cell lines grew in nude mice. Cytogenetic analysis of cell lines revealed highly aneuploid karyotypes. Deletion and/or translocation of chromosome 17 was seen in HS-16, HS-18 and HS-30 cells, and both copies of chromosome 13 were lost or re-arranged in the HS-18 cell line. Mutant p53 protein was present in all 4 cell lines. HS-18 cells showed no expression of the Rb protein and high levels of expression of E2F, cyclin A, cyclin E and CDK2. HS-16 expressed a higher level of cyclin D than the other 3 cell lines. p21WAF1 expression was seen in all cell lines, but p16ink4 was expressed only in HS-30 and HS-42 cell lines. These cell lines were sensitive to taxol and relatively resistant to methotrexate, vinblastine and 5-fluorouracil when compared with the fibrosarcoma cell line HT-1080. These new cell lines should provide a useful model for the study of soft tissue sarcomas and for evaluating new drugs or treatments.

Animals↗
PubMed1996

Calcium-sensitive transcriptional activation of the proximal CCAAT regulatory element of the grp78/BiP promoter by the human nuclear factor CBF/NF-Y.

Transcription of the gene encoding GRP78/BiP, a calcium-binding molecular chaperone localized in the endoplasmic reticulum, is induced in mammalian cells through gradual depletion of the intracellular calcium stores. The multimeric CCAAT binding factor, CBF/NF-Y, binds to the most proximal CCAAT regulatory element (C1) of the grp78 promoter required for both basal level expression and stress response. Using an in vitro transcription system, we show through factor competition and immunodepletion that the grp78 C1-mediated enhancement of transcription requires primarily CBF. Correlating with the previous observation that CBF binding to the 78C1 site is enhanced by EGTA and EDTA, these divalent cation chelators specifically stimulate 78C1-directed transcription. In contrast, increasing amounts of calcium ions are inhibitory. These results provide evidence that CBF is functionally important in transactivating the grp78 C1 transcriptional activity, and suggest a possible mechanism by which grp78 transcription is stimulated by calcium depletion. We further discovered that in addition to binding CBF, both the 78C1 element and the CBF binding site of the alpha2(I) collagen promoter interact weakly with the multifunctional transcription factor YY1. Our studies show that the binding sites for CBF and YY1 are distinct for the two promoter sites, suggesting that YY1 and other interacting factors could exert differential effects on individual promoters bearing the same CBF site.

Binding Sites↗
PubMed1996

The tumor promoter arsenite stimulates AP-1 activity by inhibiting a JNK phosphatase.

Trivalent arsenic (As3+) is highly carcinogenic, but devoid of known mutagenic activity. Therefore, it is likely to act as a tumor promoter. To understand the molecular basis for the tumor-promoting activity of As3+, we examined its effect on transcription factor AP-1, whose activity is stimulated by several other tumor promoters. We found that As3+, but not As5+, which is toxic but not carcinogenic, is a potent stimulator of AP-1 transcriptional activity and an efficient inducer of c-fos and c-jun gene expression. Induction of c-jun and c-fos transcription by As3+ correlates with activation of Jun kinases (JNKs) and p38/Mpk2, which phosphorylate transcription factors that activate these immediate early genes. No effect on ERK activity was observed. As5+, on the other hand, had a negligible effect on JNK or p38/Mpk2 activity. Biochemical analysis and co-transfection experiments strongly suggest that the primary mechanism by which As3+ stimulates JNK activity involves the inhibition of a constitutive dual-specificity JNK phosphatase. This phosphatase activity appears to be responsible for maintaining low basal JNK activity in non-stimulated cells and its inhibition may lead to tumor promotion through induction of proto-oncogenes such as c-jun and c-fos, and stimulation of AP-1 activity. The same phosphatase may also regulate p38/Mpk2 activity.

Arsenites↗
PubMed1996

Effect of cyclin D1 overexpression on drug sensitivity in a human fibrosarcoma cell line.

BACKGROUND: Alterations in the expression of genes that control the cell cycle may be of critical importance in determining the sensitivity of cells and tumors to drugs (chemosensitivity) and radiation. Mutations and deletions of the p53 tumor suppressor gene in cell lines and tumors are associated with resistance to a variety of DNA-damaging agents. The effects of alterations in the cyclin genes and their products on drug action have not been studied. One of these genes, cyclin D1, is expressed in early G1 phase, and its protein product, together with the cyclin-dependent kinases CDK4 and CDK6, mediates the phosphorylation and functional inactivation of the retinoblastoma protein (pRb). Elevated levels of expression of cyclin D1 protein have been found in a variety of cancers, including breast cancer, head and neck cancer, non-small-cell lung cancer, and mantle cell lymphomas. PURPOSE: This study was conducted to investigate the effect of increased expression of cyclin D1 protein on the chemosensitivity profile of a human fibrosarcoma cell line. METHODS: Expression plasmids containing either the neomycin-resistance gene and the complementary DNA sequence encoding human cyclin D1 or the neomycin-resistance gene only (control) were transfected by lipofection into the human HT1080 fibrosarcoma cell line, and cell colonies resistant to the antibiotic neomycin (G418) were isolated. Cyclin D1 messenger RNA (mRNA) and protein levels were measured by ribonuclease protection and western blot analyses, respectively. Dihydrofolate reductase (DHFR) mRNA and protein levels were measured by northern blot and western blot analyses, respectively. The phosphorylation status of pRb was assessed by western blot analysis. Cell cycle analysis was performed by use of the technique of fluorescence-activated cell sorting. Cytotoxicity assays were carried out by use of the sulforhodamine blue assay. RESULTS: Of the 16 cyclin D1-transfected cell clones that were isolated, four were randomly selected for further study. Two cell clones expressed high levels of cyclin D1 mRNA and protein as compared with control cells transfected with plasmids containing the neomycin-resistance gene only. A relative increase in the phosphorylated form of pRb in cells expressing high versus low levels of cyclin D1 was also revealed by western blot analysis. There was an increased fraction of cells in the S and G2 phases of the cell cycle among cells expressing higher levels of cyclin D1. Transfectants with increased cyclin D1 expression also had increased DHFR mRNA and protein expression. Cytotoxicity assays revealed a statistically significant (P < .01) increase in resistance to methotrexate in cells expressing high levels of cyclin D1 compared with cells expressing lower levels. There was no difference in resistance to doxorubicin, paclitaxel (Taxol), and cytarabine. CONCLUSION: Alterations in the expression of cyclin D1 led to altered cell cycle distribution in a human sarcoma cell line. The associated increase in DHFR expression resulted in increased resistance to methotrexate but had no effect on other classes of anticancer agents. IMPLICATIONS: These results indicate that alterations in cell cycle genes may differ in their effects on cytotoxicity. It will be important to determine the effects of alterations of other cell cycle regulatory genes on the responses of cells to specific classes of drugs. Tumors with overexpression of cyclin D1 may be relatively refractory to methotrexate treatment.

Antimetabolites, Antineoplastic↗
PubMed1996

Resistance mechanisms to methotrexate in tumors.

The mechanisms of intrinsic and acquired resistance to methotrexate (MTX) in human tumors are reviewed herein. In blasts from patients with acute lymphocytic leukemia, resistance mechanisms found are decreased uptake and increased dihydrofolate reductase (DHFR) activity. A major cause of intrinsic resistance to MTX in soft tissue sarcoma cells and in acute myelocytic leukemia appears to be a lack of drug retention, due mainly to low levels of polyglutamylation. A novel association between lack of the retinoblastoma protein and intrinsic MTX resistance has been found. This has been attributed to an increase in DHFR activity, due to an increased rate of transcription of this gene, stimulated by an increase in levels of free E2F, not sequestered by hypophosphorylated retinoblastoma protein.

Antimetabolites, Antineoplastic↗
PubMed1996

Immunohistochemical studies of GABA and parvalbumin in the developing human cerebellum.

The localization of GABA and parvalbumin was studied in the developing cerebellum of human fetuses from 16 to 28 weeks of gestation. The avidin-biotin complex immunohistochemical method combined with silver staining were used to reveal the presence of GABA- and parvalbumin-positive neurons and nerve fibres. As early as the 16th week of gestation, GABA immunopositivity was observed in the cerebellar cortex and the deep nuclei. GABA-positive neurons included Purkinje cells, stellate and basket cells of the cerebellar cortex and neurons in the deep nuclei. The gradient of immunoreactivity increased with the maturing cells, being weak at 16 weeks and becoming markedly pronounced at 28 weeks of gestation. GABA-immunopositive mossy fibres were observed in the granular cell layer at 16 weeks, and by 28 weeks, a robust fibre network was present in the cortex and deep nuclei. Immunohistochemical localization for parvalbumin indicates that weak immunoreactivity was observed in Purkinje cells, stellate and basket cells at 16 weeks of gestation, increasing in intensity with advancing age, notably in the Purkinje cells which had acquired an elaborate arbor of neurites at 28 weeks of gestation. In the deep nuclei, parvalbumin-positive cells and nerve fibres were observed throughout the 16 to 28 week period. These results indicate that GABA- and parvalbumin-positive neurons and fibres appeared as early as 16 weeks of gestation, expressing a high degree of immunoreactivity by the 28 week of fetal age.

Cerebellum↗