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Biomedical subjects

S K Kong

Publications and source records attributed to S K Kong.

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

Inhibition of glucose transporter gene expression by antisense nucleic acids in HL-60 leukemia cells.

Glucose is the basic source of energy for mammalian cells. The energy-independent transport of glucose down its concentration gradient is mediated by the facilitative glucose transporter family (GLUT). It has long been recognised that glucose transporter genes are overexpressed in many human cancer cells, to help provide extra energy for the rapid growth of cancer cells. In the present study, antisense oligonucleotides and plasmid-derived antisense RNA against GLUT-1 gene were synthesized and transfected into human leukemia HL-60 cells to investigate the effect of these antisense nucleic acids on tumour growth. Our results show that antisense nucleic acids inhibited the proliferation of HL-60 cells by 50-60% and the mRNA expression of GLUT-1 gene was suppressed as detected by Northern hybridization.

Blotting, Northern↗
PubMed1999

Enhanced cytotoxicity and suppression of glucose transport rate by combined treatment of recombinant human tumour necrosis factor-alpha and hyperthermia on L929 cells.

Combined treatment with human recombinant TNF-alpha (rhTNF-alpha) and hyperthermia at 43 degrees C arrested the growth of mouse fibrosarcoma L929 cells in vitro. The cytotoxic effect was enhanced in combined treatment compared with that following administration of rhTNF-alpha or hyperthermia alone. When the cells were subjected to hyperthermia at 43 degrees C for 3 hours and then incubated with 0.4 ng/ml rhTNF-alpha at 37 degrees C for 24 hours, a statistically significant 65% decrease in the rate of cellular glucose uptake was observed. This suppressive effect was synergistic in terms of effect achieved by rhTNF-alpha or hyperthermia individually. Since the growth of tumour cells depends mainly on catabolism of glucose, our findings indicate that one manner by which combined rhTNF-alpha and hyperthermia treatment inhibits L929 cell growth may be by reducing the supply of glucose to the cells.

Animals↗
PubMed1999

Inhibition of glucose uptake and suppression of glucose transporter 1 mRNA expression in L929 cells by tumour necrosis factor-alpha.

Recombinant human tumour necrosis factor-alpha (rhTNF-alpha) arrested the growth and suppressed glucose uptake of mouse fibrosarcoma L929 cells in vitro. When the cells were treated with rhTNF-alpha for 24 hours, the mRNA level of glucose transporter 1 (GLUT 1), which is the only GLUT found to be present in L929 cells in our study, was suppressed in a dose-dependent manner. Since the growth of tumour cells depends mainly on glucose catabolism, our findings may indicate that rhTNF-alpha inhibits L929 cells growth by lowering the glucose transport through suppression of GLUT 1 mRNA expression in the cells.

Animals↗
PubMed1998

Ca2+ is released from the nuclear tubular structure into nucleoplasm in C6 glioma cells after stimulation with phorbol ester.

It is well established that cellular Ca2+ is an important messenger that controls many nuclear functions but the source of nuclear Ca2+ is far from clear. It has long been thought that Ca2+ is translocated from the cytosol over a long distance to activate the nuclear transcription machinery. However, this model is at best an incomplete one. With the aid of confocal microscopy, we observed tubules extended deep inside the nucleus of C6 cells in agreement with previous studies (Fricker et al. (1997) J. Cell Biol. 136, 531-544). When cells were stimulated with phorbol 12-myristate 13-acetate or phorbol 12,13-diacetate, Ca2+ was released from these tubules. DiOC6(3), a vital marker for intracellular membranes, stained the tubule in the nucleus of the same cell used for Ca2+ imaging. Moreover, results from labelling the cells with rhodamine 123 further indicate that the tubule was formed by a double-membraned invagination with mitochondria inside. Studies with acridine orange showed that chromatin was excluded from the tubules. Taken together, our results demonstrate that the nuclear tubule is a structural entity responsible for the release of Ca2+ into the nucleoplasm after stimulation with phorbol ester.

Calcium↗
PubMed1998

The nucleus of HeLa cell contains tubular structures for Ca2+ signalling.

It has long been assumed that Ca2+ are translocated from the cytosol to the cell nucleus by a long distance to activate transcription machinery buried deep in the nucleoplasm. However, this model has been recently challenged. When HeLa cells were loaded with fluo-3, highly fluorescent spots of approximately 2 microns in diameter were observed in the cell nucleus while the fluo-3 signals were low in their neighbouring nucleoplasm as determined by confocal microscopy. These fluorescent spots were devoid of but usually associated with chromatin on their boundary. When cells were stimulated by ionomycin (1 microM), the fluo-3 fluorescence in these spots increased faster than that in their neighbouring nucleoplasm. In another experiment, optical sections with hot spot(s) were used to construct 3-D images to study the morphology of the hot spots. Views of reconstruction from different angles indicated that the hot spots formed a tubular structure with a connection to the nucleocytoplasmic interface. Moreover, injection of calcium green-dextran (70 kDa), a Ca(2+)-sensitive indicator conjugated with an inert molecule of large molecular size, into the cytosol leads to a formation of signals also in a tubular shape inside the nucleoplasm. This suggests that the 'channels' are real inside the nucleus and they are derived from an invagination of the double-membraned nuclear envelope. Taken together, our results indicate (1) tubular structures are found inside the cell nucleus; (2) they are extended from the cytosol into the nucleus through the invagination of the double membraned nuclear envelope; (3) molecules of molecular size up to 70 kDa could penetrate into these 'tunnels'; (4) Ca2+ can be released or transported into the cell nucleus through these tubular structures after ionomycin stimulation; and (5) the structures are usually associated with chromatin.

Aniline Compounds↗
PubMed1998

The nuclear envelope of resting C6 glioma cells is able to release and uptake Ca2+ in the absence of chemical stimulation.

Many agonists evoke events in the cell nucleus through the control of Ca2+ signals. Recent studies using isolated nuclei have indicated that the nuclear envelope is a store for nuclear Ca2+. However, the release of Ca2+ directly from the nuclear envelope in living cells has never been reported. In the present study, we have investigated the changes of Ca2+ signals at the cyto-nucleoplasmic interface of rat C6 glioma cells using confocal microscopy. Digital imaging indicates that fluo-3, a Ca2+-sensitive fluorescent probe, was concentrated in or around the nuclear envelope. Our experiments also revealed that C6 cells at rest produced spontaneous Ca2+ spikes in the absence of chemical stimulation. The amplitude of the repetitive Ca2+ spikes was higher at the nuclear envelope than in the whole cell or cytosol. After image subtraction, circular rims of Ca2+ release and uptake were seen at the outer boundary of the nucleus. When the cells were treated with thapsigargin (2 muM), a specific Ca2+-ATPase inhibitor, a long-lasting Ca2+ release was observed at the nuclear envelope. Moreover, most of the released Ca2+ was directed inwardly to the nucleoplasm with little outward diffusion. Our results thus indicate: (1) that the nuclear envelope is a Ca2+ store that possesses the ability to discharge and sequestrate Ca2+; and (2) the Ca2+-releasing channels are present in the inner nuclear membrane.

Adenosine Triphosphate↗
PubMed1998

The rise of nuclear and cytosolic Ca2+ can be uncoupled in HeLa cells.

It has long been assumed that ionized Ca2+ is transmitted from the cytosol to the cell nucleus through the nuclear pore complexes to trigger many nuclear functions such as gene transcription after agonist stimulation. However, this hypothesis has been challenged recently. In the present study, we have investigated the interplay of Ca2+ in the cytosol and nucleus of HeLa cells upon histamine stimulation by using confocal microscopy. In resting cells, addition of histamine (50 microM) produced synchronous Ca2+ signals in the cytosol and nucleus with a stronger fluo-3 emission in the nucleus. Our results also demonstrate that the rise of cytosolic and nuclear free Ca2+ can be uncoupled after histamine stimulation. This assertion is supported by the following observations: (1) when heparin was injected into the cytosol, the increase in fluo-3 fluorescence in the cytosol mediated by histamine was almost eliminated while that in the nucleus still occurred; (2) when heparin was in the nucleus, the increase in the free nucleoplasmic Ca2+ concentration ([Ca2+]n) elicited by histamine was abolished while the rise of cytosolic Ca2+ level ([Ca2+]c) was seen; (3) when we injected inositol 1,4,5-trisphosphate (IP3) directly into the nucleus, [Ca2+]n increased; and (4) in the cells given cytosolic injections of BAPTA-dextran (70 kDa), histamine evoked additional Ca2+ transients in the nucleus. These results suggest that the nucleus of the HeLa cell has its own IP3-sensitive Ca2+ store and that the Ca2+ signals in the nucleus and cytosol can be uncoupled.

Biological Transport↗
PubMed1997

Evaluation of sunscreens with various sun protection factors in a new transgenic mouse model of cutaneous photoaging that measures elastin promoter activation.

BACKGROUND: Long-term sun exposure can cause major alterations in the papillary dermis, resulting in the deposition of massive amounts of abnormal elastic material, termed solar elastosis. Previous work has demonstrated that this type of photodamage is accompanied by an increase in elastin and fibrillin messenger RNAs and elastin promoter activity. OBJECTIVE: Our purpose was to develop a rapid and sensitive in vivo method for evaluating compounds offering protection against cutaneous photodamage. METHODS: Using a line of transgenic mice that expresses the human elastin promoter linked to a chloramphenicol acetyltransferase reporter gene, we applied sunscreens with various sun protection factors to 5-day-old mice followed by 30 minimal erythema doses of solar simulating radiation for three consecutive days. RESULTS: Solar simulating radiation alone resulted in a fivefold increase in elastin promoter activity. Sun protection factors of 2, 4, 8, and 15 yielded a reduction in promoter activity by 2.8%, 42.5%, 58.1%, and 70.3%, respectively. CONCLUSION: These results confirm the use of this system as a rapid and sensitive in vivo model for evaluating compounds that protect against photodamage.

Animals↗
PubMed1996

Peroxynitrite disables the tyrosine phosphorylation regulatory mechanism: Lymphocyte-specific tyrosine kinase fails to phosphorylate nitrated cdc2(6-20)NH2 peptide.

To determine if nitration of tyrosine residues by peroxynitrite (PN), which can be generated endogenously, can disrupt the phosphorylation of tyrosine residues in proteins involved in cell signaling networks, we studied the effect of PN-promoted nitration of tyrosine residues in a pentadecameric peptide, cdc2(6-20)NH2, on the ability of the peptide to be phosphorylated. cdc2(6-20)NH2 corresponds to the tyrosine phosphorylation site of p34cdc2 kinase, which is phosphorylated by lck kinase (lymphocyte-specific tyrosine kinase, p56lck). PN nitrates both Tyr-15 and Tyr-19 of the peptide in phosphate buffer (pH 7.5) at 37 degrees C. Nitration of Tyr-15. which is the phosphorylated amino acid residue, inhibits completely the phosphorylation of the peptide. The nitration reaction is enhanced by either Fe(III)EDTA or Cu(II)-Zn(II)-superoxide dismutase (Cu,Zn-SOD). The kinetic data are consistent with the view that reactions of Fe(111)EDTA or Cu,Zn-SOD with the cis form of PN yield complexes in which PN decomposes more slowly to form N02+, the nitrating agent. Thus, the nitration efficiency of PN is enhanced. These results are discussed from the point of view that PN-promoted nitration will result in permanent impairment of cyclic cascades that control signal transduction processes and regulate cell cycles.

Amino Acid Sequence↗
PubMed1996

Nuclear envelope acts as a calcium barrier in C6 glioma cells.

The temporal and spatial changes of intracellular free calcium ([Ca2+]i) within the cytosol and nucleis of C6 glioma cells have been investigated with laser confocal scanning microscopy to evaluate the current view that Ca2+ ions pass freely through nuclear pores by diffusion. Our results indicate that localized cytosolic Ca2+ release, which appeared as puffs, spread with an apparent diffusion rate of 0.35 +/- 0.07 microns/sec (n = 44). This release was followed by an immediate Ca2+ uptake at the resting stage. Following the treatment with thapsigargin, an inhibitor of microsomal Ca(2+)-ATPase, release of nuclear Ca2+ from certain nuclear hot zones and nuclear envelope was obtained. Most of the nuclear Ca2+ released were confined to the nuclear boundary, but a slow migration of Ca2+ towards the cytosol was observed. The apparent diffusion rate of this Ca2+ release is 0.015 microns/sec. By contrast, the inward spread into nucleus occurred with a diffusion rate of 0.04 microns/sec. From these diffusion rates and other experimental evidence, we conclude that the movement of Ca2+ at the nucleocytosolic interface is more than a simple diffusion process and the interface is a barrier to Ca2+ movement.

Animals↗
PubMed1996

K1 and K3 capsular antigens of Klebsiella induce tumor necrosis factor activities.

Capsular polysaccharide antigens isolated from Klebsiella pneumoniae sero-type 1 (K1) and sero-type 3 (K3) could induce tumor necrosis factor-alpha in ICR mice. K1 and K3 capsular antigens were found to be non-toxic by brine shrimp bioassay. When injected into Ehrlich ascites tumor-bearing mice, both K1 and K3 capsular antigens exhibited significant suppression in the growth of tumor cells. The significance of these observations is discussed.

Animals↗
PubMed1996

8-methoxypsoralen and ultraviolet a radiation activate the human elastin promoter in transgenic mice: in vivo and in vitro evidence for gene induction.

Treatment of skin diseases with the combination of 8-methoxypsoralen and ultraviolet A radiation (PUVA) results in clinical alterations in treated skin that resemble those observed in chronically photodamaged skin. The PUVA-treated patients develop nonmelanoma skin cancers, pigmentary alterations and wrinkling characteristic of sun-induced changes. The major alteration in the dermis of sun-damaged skin is the deposition of abnormal elastic fibers, termed solar elastosis. Up-regulation of elastin promoter activity in dermal fibroblasts explains the excess elastic tissue but not the reason for the aberrant morphology of the elastotic material. In order to study photoaging in an experimental system, we utilized a transgenic mouse line that expresses the human elastin promoter/chloramphenicol acetyltransferase construct in a tissue-specific and developmentally regulated manner. Although UVB radiation has been demonstrated to increase promoter activity in vitro, UVA fails to demonstrate a similar effect at the doses utilized. In this study, we demonstrate the ability of PUVA treatment to up-regulate elastin promoter activity both in vitro and in vivo. These data help to explain the development of photoaging in sun-protected PUVA-treated skin. We attribute the up-regulation of elastin promoter activity in response to PUVA to the formation of DNA photoadducts, which do not occur in response to UVA radiation alone.

Animals↗
PubMed1996

Concanavalin A-induced apoptosis in murine macrophages through a Ca(2+)- independent pathway.

Concanavalin A (ConA), normally a mitogen of T lymphocytes, was found to induce apoptosis or programmed cell death in murine peritoneal macrophages. The following observations support this assertion: 1) incubation of peritoneal macrophages or cultured PU5-1.8 macrophage cells with ConA caused a dose- and time-dependent reduction of mitochondrial dehy-drogenase activity as measured by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, 2) treatment of cells with ConA induced formation of apoptotic bodies as seen under the confocal laser scanning microscope, 3) challenge of cells with ConA produced a considerable amount of cell debris with DNA content next to G0 phase as revealed by flow cytometry and 4) ConA was able to elicit DNA fragmentation in these cells. The involvement of Ca(2+) in mediating the apoptosis was studied in single cells by confocal laser scanning microscope using the Ca(2+) fluorescence dye, fluo-3. Our results show that ConA induced an immediate rise of intracellular free Ca(2+) concentration as well as opening of Ca(2+) channels on cell surface. But when the cells were treated with 1,2-bis(o-aminophenoxy) ethane-N, N, N', N'-tetraacetic acid/AM (BAPTA/AM), a Ca(2+) chelator, to buffer the rise of internal Ca(2+), ConA still caused DNA fragmentation. Furthermore, injection of Ca(2+) into the cell with ionomycin had no stimulatory effect on DNA fragmentation. These results suggest that Ca(2+) changes induced by ConA are not a prerequisite for apoptosis in macrophages.

Journal Article↗
PubMed1994

Purification and characterization of a ubiquitin carrier protein kinase from HeLa cells.

Protein ubiquitination plays an important role in ATP-dependent protein turnover, and it also may regulate other cellular events. Covalent attachment of ubiquitin to other proteins is catalyzed by three different enzymes, E1, E2, and E3. We have previously shown that protein ubiquitination can be regulated by phosphorylation. In the present study, we show that 20-kDa E2, an E2 molecular mass isoform, is phosphorylated by a protein kinase from the cytosolic fraction of HeLa cells. This protein kinase was purified by a procedure involving ammonium sulfate precipitation and three column chromatographies (phenyl-Sepharose, Superose gel filtration, and DEAE-Sephacel). Gel-filtration chromatography indicated that the molecular mass of this protein kinase was about 300 kDa. However, SDS/PAGE showed that the purified protein kinase consists of three subunits with molecular masses of 120, 105, and 70 kDa, respectively. The stoichiometry of the phosphorylated 20-kDa E2 isozyme was found to be 0.45 mol of phosphate per mol of protein. The phosphorylation of 20-kDa E2 occurred only at the serine residue. The activity of this protein kinase required the presence of Mg2+; however, the enzyme was inhibited by a high concentration of Mg2+.

HeLa Cells↗
PubMed1994

The nitric oxide donor, sodium nitroprusside, increased intranuclear and cytosolic free calcium concentration in single PU5-1.8 cells.

In the present study, the effect of sodium nitroprusside (SNP), a nitric oxide (NO) releasing agent, on the intracellular free calcium concentration ([Ca2+]i) was studied. Our results demonstrate that SNP could increase [Ca2+]i in single PU5-1.8 cells, a murine macrophage cell line, as determined by confocal laser scanning microscope with calcium green-1 fluorescent probe. In the presence of hemoglobin, a NO scavenger, the effect of SNP on the Ca2+ level was suppressed. Our data also show that the intranuclear Ca2+ ([Ca2+]n) was always higher than those in cytosol. Stimulation of cells with 1mM SNP induced an immediate increase of [Ca2+]n with a delayed rise of cytosolic Ca2+ ([Ca2+]c). Interestingly, when cells were subsequently treated with 2mM SNP, an increase of [Ca2+]c with a similar pattern to the rise [Ca2+]n was observed. In Ca(2+)-free buffer, SNP also raised the [Ca2+]n and [Ca2+]c. When Ca2+ was added to the buffer, a second peak of [Ca2+]n and several spikes of [Ca2+]c were observed. These results thus indicate that 1) NO may increase both [Ca2+]n and [Ca2+]c in PU5-1.8 cells; 2) the increase of [Ca2+]n requires both internal and external Ca2+. Moreover, the diverse responses in these cells suggest that the regulation of Ca2+ in these areas may be different.

Animals↗
PubMed1993

Change of membrane potential is not required for the tumour necrosis factor-alpha mediated cytotoxicity.

Recent evidence has been demonstrated that trimeric tumour necrosis factor-alpha (TNF) itself is a channel forming molecule and incubation of TNF with TNF-sensitive tumour cell line U937 increases the plasmalemmal Na+ permeability (21). Therefore, membrane potential (Em) may play a role in the cytotoxicity mediated by TNF. Results in our study demonstrate that: 1) TNF did not induce any changes of Em of L-929 cells but gramicidin and valinomycin produced positive responses as measured by two fluorescent probes, bis-oxonol and dis-C3-(5). 2) Exogenously imposed membrane depolarization by gramicidin or high potassium buffer did not attenuate the TNF-mediated cytotoxicity in L929 cells. 3) When the Em of L-cells was clamped at K(+)-equilibrium potential, TNF showed a dose-dependent cytotoxicity (determined by cell morphology and neutral red uptake assay) which was very similar to the normal TNF cytotoxic action. Taken together, evidence in our study illustrates that membrane depolarization or hyperpolarization is not required for TNF-mediated cytotoxicity.

Animals↗
PubMed1992

Protein ubiquitination is regulated by phosphorylation. An in vitro study.

Protein ubiquitination has been implicated in ATP-dependent protein turnover and in a number of biological processes in eukaryotic cells. The ubiquitination activating enzyme, E1, and ubiquitin carrier protein, E2, are two essential enzymes in the protein ubiquitination machinery. Using purified E1 and E2 from rabbit reticulocytes and various protein kinases, which include cAMP-dependent protein kinase, protein kinase C, and protein tyrosine kinase, we demonstrated that E1 is phosphorylated by protein kinase C, with a stoichiometry of 0.65 mol of phosphate/mol of E1, and one of the E2 isoforms, E2(32kDa), is phosphorylated by protein tyrosine kinase to 2 eq of phosphate/mol of protein. Phosphorylation of E1 causes a 2-fold enhancement of its activity as monitored by ubiquitin-dependent ATP in equilibrium PPi exchange. When 1 eq of phosphate was incorporated into E2(32kDa), a 2.4-fold activation was also observed for its activity to catalyze the ubiquitination of histone H2A. The regulatory significance of this finding is discussed.

Adenosine Triphosphate↗