Biomedical subjects

J M Phang

Publications and source records attributed to J M Phang.

At least 19 recordsLinked to original sources

PubMed1995

Chemoprevention of spontaneous tumorigenesis in p53-knockout mice.

Spontaneous tumorigenesis was evaluated in male p53-knockout (p53-/-) mice treated with dehydroepiandrosterone (DHEA), quercetin, d-limonene, or all-trans retinoic acid to determine whether tumor development in these mice can be modulated by cancer-chemopreventive agents. DHEA-treated mice experienced a delay in tumorigenesis (particularly lymphomas) and subsequent mortality (P < 0.01) relative to untreated control mice. Quercetin, d-limonene, and all-trans retinoic acid each had no effect on spontaneous tumor development in p53-/- mice. These data demonstrate that tumor development in p53-/- mice can be delayed by DHEA and suggest that p53-/- mice provide a useful model for evaluating strategies to offset the increased risk of tumorigenesis resulting from loss of p53 tumor suppressor function.

Animals↗

PubMed1995

Effects of estrogen on apoptotic pathways in human breast cancer cell line MCF-7.

We utilized a reverse transcription-PCR method to examine the effect of estrogen on the expression of mRNA for Bcl-2 and Bax, two modulatory proteins in the apoptotic pathway, in human breast cancer cell line MCF-7. We found that the bcl-2 mRNA levels in the cells exposed to 17 beta-estradiol were higher than those of control cells. Although the relative bax mRNA levels remained unchanged, the changes in bcl-2 mRNA level occurred in a time- and concentration-dependent fashion. In addition, pretreatment with 17 beta-estradiol protected MCF-7 cells from apoptosis. Our study provides evidence that responses of breast epithelial cells toward a steroid sex hormone involve regulation of the apoptotic pathway.

Apoptosis↗

PubMed1994

Accumulation of fatty alcohol in MCF-7 breast cancer cells.

The MCF-7 cell line (human breast epithelial cells) accumulates fatty alcohols. The fatty alcohols were identified as C16:0, C18:0, and C18:1 alcohols by thin-layer chromatography and gas chromatography/mass spectrometry. This accumulation of alcohols in MCF-7 was found in cultures of MCF-7 cells obtained from other laboratories but not in a variety of unrelated cell lines. The presence of the alcohols suggested an aberrant ether lipid metabolism in the MCF-7 cells. Therefore, the capacity for either lipid biosynthesis was evaluated using cells incubated with either [14C]stearyl alcohol or [14C]stearic acid. MCF-7 cells incorporated less than 0.4% of the [14C]alcohol into ether-linked phospholipids, whereas the AB589 breast epithelial cells, used as a "normal control" for comparisons, did not accumulate fatty alcohol and incorporated approximately 20% of the radiolabeled alcohol into phospholipids containing ether linkages. Although the MCF-7 cells were unable to effectively incorporate the fatty alcohol into ether linkages, the cells were able to oxidize the alcohol to fatty acid. When incubated with [14C]stearic acid, the conversion to radiolabeled fatty alcohol in MCF-7 cells was approximately four times higher than the alcohol levels found in AB589 cells. While deficient in the ability to synthesize ether linkages, the MCF-7 cells did incorporate radiolabeled hexadecylglycerol, a precursor containing an ether linkage, into phospholipids. Collectively, the data indicate that the MCF-7 cells possess a deficiency in the alkyl DHAP synthase activity. A near absence of ether-linked lipids in the MCF-7 cells was indicated by the radiolabeling studies, and this finding was corroborated by results from HPLC analysis. Analyses of the partial glycerides, obtained from the enzymatic hydrolysis of cellular phospholipids, found only trace levels of ether lipids in the MCF-7 cells. The aberration in ether lipid biosynthesis did not correlate with the expression of the multidrug resistance phenotype in a series multidrug resistant MCF-7 variants. The results are discussed relative to the use of the MCF-7 cells as a model for investigations of ether lipid biosynthesis and the cellular physiology of ether lipids.

Breast Neoplasms↗

PubMed1994

Modulation of adriamycin accumulation and efflux by flavonoids in HCT-15 colon cells. Activation of P-glycoprotein as a putative mechanism.

Since P-glycoprotein (P-gp) in normal tissues may serve as a cellular defense mechanism against naturally occurring xenobiotics, we considered whether physiologically active components of commonly ingested plant foods could influence P-gp function. To examine this possibility, a series of flavonoids commonly found in plant foods was tested for their ability to modulate [14C]Adriamycin ([14C]ADR) accumulation and efflux in P-gp-expressing HCT-15 colon cells. Many flavonoids, in the micromolar range, inhibited the accumulation of [14C]ADR. Detailed experiments utilizing flavonoids with the greatest activity in reducing [14C]ADR accumulation, i.e. galangin, kaempferol, and quercetin, revealed that the efflux of [14C]ADR is increased markedly in the presence of these compounds. Flavonoid-induced stimulation of efflux was rapid and was blocked by the multidrug-resistant (MDR) reversal agents verapamil, vinblastine, and quinidine. The magnitude of flavonoid-stimulated efflux in sodium butyrate-treated cells with a 4-fold induction of P-gp protein was similar to that in uninduced cells. [3H]Azidopine photoaffinity labeling of P-gp in crude membrane preparations revealed mild to no competition for binding by flavonoids possessing either activity or inactivity in reducing ADR accumulation. Although flavonoid hydrophobicity was found to be unrelated to flavonoid activity in altering [14C]ADR accumulation, certain structural features were associated with enhancement or diminution of activity. Finally, the significance of flavonoid-related reduction of [14C]ADR accumulation was underscored in cell growth studies, showing partial protection by quercetin against ADR-induced growth inhibition. It is concluded that certain naturally occurring plant flavonoids may acutely upregulate the apparent activity of P-gp.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

Effects of N-(4-hydroxyphenyl)retinamide supplementation on vitamin A metabolism.

The efficacy of the retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) has been demonstrated in the inhibition of cancers in a variety of tissues. Moreover, toxicity effects following administration of 4-HPR have been found to be reduced or absent when compared to other retinoids. Pharmacokinetic studies in both animals and humans have focused on the metabolism of 4-HPR and its metabolites, and relatively little information has been published detailing the effects of long-term administration of 4-HPR upon normal endogenous vitamin A metabolism. Thus, the present study was carried out to examine the effects of long-term administration of 4-HPR upon plasma and tissue vitamin A kinetics. Male Sprague-Dawley rats were fed either a control diet sufficient in vitamin A [CON group; 1.0 retinol (ROH) equivalents/g diet] or a CON diet supplemented with 4-HPR (CON+4HPR group; 1173 micrograms 4-HPR/g diet). Following i.v. injection of a physiologically radiolabeled dose of ROH, ROH tracer and tracee kinetics were monitored in plasma and tissues over a 41-day period. Kinetic parameters were determined using the SAAM/CONSAM computer modeling programs to carry out graphical analysis of the tracer concentration curves. Mean plasma ROH levels measured for the CON+4HPR group were reduced to one-third of those of the CON group. Most of the kinetic parameters calculated were found to be significantly altered by the inclusion of 4-HPR in the diet. The fraction of the plasma ROH being catabolized per day (fractional catabolic rate) was nearly twice as high in the CON+4HPR treated group (3.61 +/- 0.49 day-1; mean +/- SD) as compared to the CON group (2.00 +/- 0.68 day-1). The amount of time that vitamin A molecules spent in the body before being lost irreversibly from the system (system residence time) was decreased by half in the CON+4HPR group (19.20 +/- 7.13 days) versus the CON group (38.63 +/- 9.62 days). Despite the increased catabolic rates and decreased system residence times measured for the CON+4HPR group, the estimated vitamin A use in these animals (11.01 +/- 3.10 micrograms/day) was 33% less than that used by the CON group (16.31 +/- 2.47 micrograms/day). Studies investigating the mechanisms by which 4-HPR alters vitamin A kinetics are presently under way in our laboratory. Nevertheless, these results suggest that long-term administration of 4-HPR markedly perturbs normal vitamin A metabolism in rats. Whether 4-HPR similarly alters human vitamin A metabolism with untoward clinical consequences deserves careful evaluation.

Animals↗

PubMed1994

Calorie restriction delays spontaneous tumorigenesis in p53-knockout transgenic mice.

Transgenic mice with both alleles of the p53 tumor suppressor gene (frequently mutated in human tumors) knocked out by gene targeting provide a potentially useful tumorigenesis model because these mice rapidly develop spontaneous tumors. To determine whether tumorigenesis in p53-knockout mice is sensitive to experimental manipulation, tumor development in response to calorie restriction (CR; a potent inhibitor of rodent tumors) was evaluated. Tumor development was monitored for 48 weeks in male nullizygous p53-knockout and wild-type littermate mice (28-30 per treatment group) fed ad libitum (AL) or restricted to 60% of AL carbohydrate calorie intake. CR:p53-knockout mice (median survival = 25 weeks) experienced a delay in tumor onset and subsequent mortality (P = 0.0002) relative to AL:p53-knockout mice (median survival = 16 weeks). Tumor development and mortality in wild-type littermates on either diet treatment were < 4% through 48 weeks. Cell cycle analyses were performed on splenocytes from p53-knockout mice and wild-type littermates after 4 weeks of AL feeding or CR (5 per group). The percentage of splenocytes in S phase of the cell cycle was 3-fold higher for p53-knockout mice than wild-type mice (P < 0.001), and CR reduced the percentage of S-phase splenocytes in both p53-knockout and wild-type mice (P = 0.012). These data demonstrate that tumor development in p53-knockout mice genetically predisposed to tumors can be delayed by CR (possibly via cell cycle modulation) and suggest that these mice provide a very useful model of spontaneous tumorigenesis.

Alleles↗

PubMed1994

Increased phospholipase D activity in multidrug resistant breast cancer cells.

An adriamycin-resistant MCF-7 cell line (300-fold increased resistance vs. wild type MCF-7) showed an increased phorbol ester-stimulated phospholipase D activity of approximately 4-6 times over that found in wild type cells. Phospholipase D activity was assessed by monitoring the mass of phorbol ester-induced phosphatidylethanol. The cellular phospholipase D activity was time- and phorbol ester-concentration-dependent and was obvious whether measured as an increase in the mass of PEt or in the production of 3H-labeled phosphatidylethanol in cells prelabeled with [3H]myristic acid. Phorbol ester also stimulated increases in the production of the mass of cellular diacylglycerol and phosphatidic acid in the adriamycin-resistant cells vs. the wild-type cells. Tests with a series of drug-resistant MCF-7 cell lines revealed a positive correlation between increased drug resistance and phorbol ester-stimulated phospholipase D activity.

Breast Neoplasms↗

PubMed1994

Stimulation of pS2 expression by diet-derived compounds.

Epidemiological studies suggest a lowered risk of hormone-dependent cancers among vegetarians, but the basis for this association remains unclear. Vegetables and fruits contain certain compounds which can be converted to biologically active hormone-like substances, such as lignans and isoflavones, by intestinal flora. The interaction of these compounds with endogenous hormones may be a novel, diet-dependent mechanism in cancer prevention. To explore this possibility, we developed a rapid, specific assay system to screen for compounds with estrogen-like activity in tissue culture. We utilized the estrogen receptor-positive breast cancer cell MCF-7 and monitored the expression of the estrogen-responsive protein pS2 by Northern blots. Our results indicated that the phenolic compounds daidzein, equol, nordihydroguaiaretic acid, enterolactone, and kaempferol were able to elicit an estrogen-like response, while quercetin and enterodiol were not.

Breast Neoplasms↗

PubMed1993

Flavonol-stimulated efflux of 7,12-dimethylbenz(a)anthracene in multidrug-resistant breast cancer cells.

We used a series of P-glycoprotein (P-gp) expressing multidrug-resistant (MDR) cells, developed from human breast cancer MCF-7 cells by exposure to Adriamycin, to investigate the effects of flavonoids on P-gp-mediated efflux mechanisms for chemical carcinogens. We previously showed that MDR cells derived from exposure to Adriamycin are cross-resistant to a chemical carcinogen, benzo(a)pyrene, due to its cellular efflux by the P-gp-mediated putative drug efflux pump. Our current studies extended this observation to another polycyclic aromatic hydrocarbon, 7,12-dimethylbenz(a)anthracene, known to induce mammary tumors in animals. In our attempt to find naturally occurring dietary compounds which may stimulate the P-gp-mediated efflux of carcinogens, we found that certain flavonols, kaempferol, quercetin, and galangin, are potent stimulators of the P-gp-mediated efflux of 7,12-dimethylbenz(a)-anthracene. The increased efflux decreased the cellular burden of 7,12-dimethylbenz(a)anthracene. Since these flavonol compounds are widely distributed in fruits and vegetables, their stimulatory effect on P-gp may be a mechanism relevant to carcinogenesis and the observed lowered cancer risk in humans with higher dietary intake of fruits and vegetables.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1993

Production of human plasma retinol-binding protein in Escherichia coli.

We designed a polymerase chain reaction (PCR) primer pair which allowed us to clone the cDNA coding for the human plasma retinol-binding protein (hRBP) into an Escherichia coli expression vector. Production of hRBP was confirmed by probing Western blots with antisera against plasma hRBP. Purification and characterization of the E. coli-produced plasma hRBP are also described. The availability of this expression system makes it possible to obtain large quantities of hRBP to facilitate our continuing studies of retinol and RBP metabolism.

Amino Acid Sequence↗

PubMed1993

Hydrolysis of proline dipeptides completely fulfills the proline requirement in a proline-auxotrophic Chinese hamster ovary cell line.

Proline- and hydroxyproline-containing oligopeptides may be important in protein nutrition because intestinal hydrolases are incapable of recognizing their imido bonds. Peripheral tissues have a cytosolic enzyme prolidase that cleaves dipeptides containing C-terminal proline (X-Pro) or hydroxyproline. The role of dipeptides in intracellular metabolism is uncertain. This study examined the ability of X-Pro to provide proline to the proline-auxotrophic cell line, CHO-K1. The action of prolidase on exogenously supplied Gly-Pro, the most abundant dipeptide product of digestion, provided adequate proline to support normal cell growth of CHO-K1 cells in a dose-dependent manner. The growth curve generated by addition of Gly-Pro to CHO-K1 cells was similar to that due to proline. Two other structurally unrelated X-Pro also supported growth indistinguishably from Gly-Pro. Gly-Hyp was completely ineffective for growth. The ability of X-Pro to sustain cultures of a proline-auxotrophic cell line may be important in elucidating intracellular nutritional and physiological functions for those dipeptides.

Animals↗

PubMed1992

A new functional role for P-glycoprotein: efflux pump for benzo(alpha)pyrene in human breast cancer MCF-7 cells.

We propose that the cellular burden of certain carcinogens may be mitigated by P-glycoprotein (P-gp), the putative drug efflux pump. In a series of multidrug resistant human breast cancer MCF-7 cells with increasing P-gp expression we examined this hypothesis using benzo(alpha)pyrene, a widely distributed environmental and dietary carcinogen. We found that multidrug resistant cells were cross-resistant to benzo(alpha)pyrene and the rates of efflux for benzo(alpha)pyrene were higher in multidrug resistant cells than in wild type cells. Evidence supporting the involvement of P-gp included the inhibition of azidopine binding to P-gp benzo(alpha)pyrene and the inhibition of benzo(alpha)pyrene efflux by Adriamycin and verapamil. Our findings suggest that P-gp may play a role in the cellular defense to carcinogens. The expression of P-gp and the modulation of its function may affect the susceptibility of normal tissues to transformation by carcinogens.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1991

Structural analogues of pyrroline 5-carboxylate specifically inhibit its uptake into cells.

Pyrroline 5-carboxylate, a naturally occurring intermediate, is a potent activator of redox-dependent metabolic pathways. The effect of pyrroline 5-carboxylate is due, at least in part, to the special mechanism mediating its entry into cells. Using Chinese hamster ovary cells we recently characterized the cellular uptake of pyrroline 5-carboxylate as a process transferring oxidizing potential pari passu with cell entry, a process consistent with group translocation. We sought to identify specific inhibitors to probe this unique uptake mechanism, to blockade the metabolic effects of pyrroline 5-carboxylate, and to provide strategies to identify the putative carrier protein. Because pyrroline 5-carboxylate, a ring structure with a tertiary nitrogen, is in spontaneous equilibrium with glutamic-gamma-semialdehyde, an open-chain structure, we tested analogues of both. Most open-chain aldehydes at 10 mM had little effect on the uptake of pyrroline 5-carboxylate. Although succinic semialdehyde did inhibit, its effect was nonspecific in that the uptake of alpha(methylamino) isobutyric acid was inhibited as much as the uptake of pyrroline 5-carboxylate. In contrast, pyrroline 2-carboxylate and other cyclic compounds with tertiary nitrogens, e.g., pyridines, were specific inhibitors of pyrroline 5-carboxylate uptake. Respective potencies of pyridine derivatives depended on the nature and location of constituent groups. Kinetics studies showed that these inhibitors were competitive with pyrroline 5-carboxylate and the most potent inhibitor, 2,6-pyridinedicarboxaldehyde, exhibited a K12 of 0.27 +/- 0.05 mM.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Accumulation of pyrroline 5-carboxylic acid in conditioned medium of cultured fibroblast: stimulatory effects of serum, insulin, and IGF-1.

Pyrroline 5-carboxylate, an intermediate of amino acid metabolism, is released into medium by cultured normal human fibroblasts. With cells made quiescent by serum starvation, the addition of 10% fetal bovine serum augmented the release of pyrroline 5-carboxylate into medium by 2.5-fold. Although platelet-derived growth factor was without effect, both insulin and insulinlike growth factor-1 nearly reproduced the serum effect. The dose-dependence of insulin and insulinlike growth factor 1 effects suggested their mediation by their own respective receptors. Although the mechanism for the stimulatory effect remains unknown, these effects of insulin and insulinlike growth factor 1 on pyrroline 5-carboxylate suggest hormonal regulation of pyrroline 5-carboxylate release.

Blood↗

PubMed1989

Purified human erythrocyte pyrroline-5-carboxylate reductase. Preferential oxidation of NADPH.

Pyrroline-5-carboxylate reductase catalyzes the final step in proline synthesis by NAD(P)H-dependent reduction of pyrroline-5-carboxylate. We have purified and characterized this enzyme from human erythrocytes. Purification to homogeneity (approximately 600,000-fold) was accomplished by sonication, ultracentrifugation, 2',5'-ADP-Sepharose affinity chromatography, and DEAE-Sephacel ion exchange chromatography. The enzyme runs as a single band of 30,000 Mr on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sizing chromatography under nondenaturating conditions demonstrates activity in the 300,000-350,000 Mr range, suggesting that the native enzyme exists as a 10- to 12-mer. The purified enzyme exhibits kinetic characteristics similar to those previously described for whole red cell homogenates. The Vmax is 10-fold higher and the Km for pyrroline-5-carboxylate is 7-fold higher with NADH versus NADPH as cofactor. The affinity for NADPH is 15-fold higher than that for NADH. Erythrocyte pyrroline-5-carboxylate reductase is competitively inhibited by NADP+. Unlike the enzyme from some other sources, erythrocyte pyrroline-5-carboxylate reductase is not inhibited by proline or ATP. Double label studies using [14C]pyrroline-5-carboxylate and [3H]exNADPH in the presence of both NADH and NADPH were performed to determine the preferred source of reducing equivalents. In the presence of physiologic concentrations of pyrroline-5-carboxylate and both pyridine nucleotides, all of the reducing equivalents came from NADPH. We suggest that, in some cell types including human erythrocytes, a physiologic function of pyrroline-5-carboxylate reductase is the generation of NADP+.

1-Pyrroline-5-Carboxylate Dehydrogenase↗

PubMed1989

Type II hyperprolinaemia in a pedigree of Irish travellers (nomads).

We describe a study of 312 subjects in 71 families near related to a proband with type II hyperprolinaemia. The subjects were Irish travellers (nomads) among whom consanguineous marriage and high fertility are common. Thirteen additional cases of type II hyperprolinaemia were discovered; all were offspring of consanguineous unions. A further 50 subjects were found to have mild hyperprolinaemia. We found a strong association between type II hyperprolinaemia and seizures during childhood but no significant association with mental handicap. Most adults with type II hyperprolinaemia enjoyed normal health and there was no evidence that maternal hyperprolinaemia compromised fetal development. The documented association between type II hyperprolinaemia and seizures may be related to the neuromodulatory or reducing-oxidising effects of proline and pyrroline-5-carboxylate, respectively, that has been shown in vitro. Alternatively, another genetic defect closely linked to the type II hyperprolinaemia allele could be the explanation.

1-Pyrroline-5-Carboxylate Dehydrogenase↗

PubMed1989

Fluctuations in plasma pyrroline-5-carboxylate concentrations during feeding and fasting.

Pyrroline-5-carboxylate (P5C) is the oxidized metabolite of proline. In cultured cells redox-sensitive metabolic pathways are influenced by these two amino acids. To determine whether plasma P5C levels fluctuate enough physiologically to regulate such pathways in vivo, we measured venous plasma P5C concentrations hourly for 48 h in five normal subjects while eating and in four subjects while fasting. Two fed subjects had one or more plasma P5C excursions per day exceeding 10 times the baseline level; the other fed subjects had lesser variations. These patterns suggest a relationship of plasma P5C peaks to meals. This relationship is supported by the finding of no significant P5C elevations above baseline levels in four subjects during fasting for 24 h. Despite the changes in plasma P5C concentrations, those of proline and other metabolically related amino acids were constant. The variation among subjects in the degree of plasma P5C fluctuation could not be explained by differences in total protein and caloric intake or body weight. Among plasma constituents, the large peak to basal plasma P5C excursions in some of the normal subjects we studied are matched only by those of peptide hormones.

Adult↗

PubMed1988

Stimulation of phosphoribosyl pyrophosphate and purine nucleotide production by pyrroline 5-carboxylate in human erythrocytes.

Recent studies have shown that pyrroline 5-carboxylate, the intermediate in the interconversions of proline, ornithine, and glutamate, can regulate the metabolism of erythrocytes. We now report that the formation of 5-phosphoribosyl 1-pyrophosphate (PP-Rib-P) was markedly stimulated by pyrroline 5-carboxylate in intact red cells. The production of PP-Rib-P is an important point of regulation in nucleotide metabolism. We found that pyrroline 5-carboxylate increased glucose metabolism through the oxidative arm of the pentose shunt, ribose 5-phosphate formation, and PP-Rib-P production and subsequently augmented purine nucleotide production through the salvage pathway in erythrocytes. We now report that pyrroline 5-carboxylate markedly stimulated the net synthesis of inosine monophosphate from hypoxanthine in intact human red cells so that the pool of inosine monophosphate became 20-30% of the total pool of purine nucleotides. Inosine monophosphate has been considered to be a "mobile pool" of purines, i.e. a reservoir from which peripheral tissues can be supplied; the effect of pyrroline 5-carboxylate on the inosine monophosphate pool may be a mechanism for regulating the function of erythrocytes in purine delivery.

Adenine Nucleotides↗