Search PubMed⌕ Search

Biomedical subjects

S A McCormack

Publications and source records attributed to S A McCormack.

At least 37 records · Page 2Linked to original sources
PubMed1994

Polyamines influence transglutaminase activity and cell migration in two cell lines.

Transglutaminases (TGAs) catalyze the cross-linking of proteins through formation of gamma-glutaminyl-epsilon-lysine bonds and incorporation of small-molecular-weight amines, including polyamines, into the gamma-glutamine sites of proteins. Tissue TGA has been shown to establish covalent cross-links between cytoskeletal proteins using polyamines as substrates, and protein-polyamine conjugates have been identified in a variety of cells. We have shown previously that polyamines are required for cell migration in IEC-6 cells [S. A. McCormack, M. J. Viar, and L. R. Johnson. Am. J. Physiol. 264 (Gastrointest. Liver Physiol. 27): G367-G374, 1993]. In this study, we explored the relationship between cell migration, polyamines, and tissue TGA activity in two cell lines and found that while both IEC-6 and Caco-2 cells required normal levels of polyamines to migrate across a denuded surface, tissue TGA activity responded differently to polyamine deficiency brought about by treatment with alpha-difluoromethylornithine (DFMO). DFMO is a specific and irreversible inhibitor of ornithine decarboxylase, a rate-limiting enzyme of polyamine biosynthesis. In IEC-6 cells, tissue TGA activity decreased significantly with DFMO treatment concurrent with a rise in inactive TGA protein as measured by Western blot analysis. On the other hand, in Caco-2 cells, tissue TGA activity and protein increased significantly with DFMO treatment. In both cell lines, addition of polyamines to the DFMO treatment restored cell migration, tissue TGA activity, and protein to control levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Polyamine deficiency causes reorganization of F-actin and tropomyosin in IEC-6 cells.

In earlier work we have shown that polyamine-deficient IEC-6 cells lose most of their ability to migrate. In this report we describe the effect of polyamine deficiency on the cytoskeleton of migrating IEC-6 cells. Cells were grown on cover slips for 4 days. One-third of the monolayer was removed, and the remainder was incubated for 6 h. The monolayers were fixed and stained with rhodamine phalloidin for actin filaments and by immunocytochemistry for tropomyosin. In control cells, actin filaments were found as stress fibers traversing the cell, in a thin actin cortex often visible on only one edge of the cell, and in fine fibers extending into the lamellipodia. Tropomyosin was found in the same distribution. A Western blot showed that tropomyosin was present as 35- and 37-kDa isoforms. In polyamine-deficient cells, actin stress fibers were less dense, whereas the actin cortex was greatly increased in density and lamellipodia were less extensive. Tropomyosin distribution was similar and included a 30-kDa isoform not seen previously. In spite of the obvious changes in the distribution of these cytoskeletal proteins, the concentrations of filamentous actin, beta-actin mRNA, and the higher molecular weight tropomyosin isoforms did not change. In all cases the addition of putrescine to polyamine-deficient cells prevented the changes described. We conclude that polyamines are essential for migration in this system because of their effects on the organization of cytoskeletal actin, tropomyosin, and perhaps other proteins as well.

Actins↗
PubMed1994

Secretin inhibits induction of ornithine decarboxylase activity by gastrin in duodenal mucosa and IEC-6 cells.

Ornithine decarboxylase (ODC) catalyzes the first rate-limiting step in polyamine biosynthesis, and increased ODC activity is one of the earliest biochemical events associated with the induction of cellular proliferation. The current study examines the regulation of ODC activity in rat duodenal mucosa and IEC-6 cells (a line of normal rat intestinal crypt cells) in response to the trophic hormone, gastrin, and its inhibitor, secretin. Rats were fasted 22 h before the various treatments, and ODC activity was measured in scraped duodenal mucosa. Gastrin significantly increased ODC activity within 3 h to 4.3 times control levels. The effect of gastrin was totally inhibited by 5 micrograms/kg secretin. In doses of 5 or 10 micrograms/kg, secretin had no effect on basal ODC. Epidermal growth factor (EGF) and refeeding fasted rats also significantly increased ODC activity in duodenal mucosa, but the effects of EGF and refeeding were not prevented by secretin. In cultured IEC-6 cells, ODC activity was significantly increased after exposure to gastrin, 5% dialyzed fetal bovine serum (FBS), EGF, and asparagine. Secretin in doses ranging from 10(-10) to 10(-6) M caused a linear and significant inhibition of the stimulation of ODC activity by gastrin. No dose of secretin affected basal ODC activity or enzyme activity stimulated by 5% dialyzed FBS, EGF, or asparagine in IEC-6 cells. The ODC mRNA levels in IEC-6 cells were also increased after exposure to gastrin. Administration of secretin significantly prevented the stimulated expression of the ODC gene in cells treated with gastrin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Polyamines are necessary for cell migration by a small intestinal crypt cell line.

Studies from our laboratory have shown that polyamines are essential for the normal repair of duodenal erosions induced in vivo in a rat stress-ulcer model. In that model, the inhibition of ornithine decarboxylase, a rate-limiting enzyme of polyamine biosynthesis, with alpha-difluoromethylornithine (DFMO) almost entirely prevented healing. Healing could be restored by oral polyamines. In this paper, we have investigated whether the polyamines are required for the early stages of epithelial restitution using an IEC-6 cell culture model of cell migration. Treatment of the cells with DFMO for 4 days reduced cell migration 80%. Migration could be restored to normal by concomitant treatment with putrescine (PUT), spermidine (SPD), or spermine (SPM), but not by their addition during the migration period (6 h) only. If DFMO treatment was not begun until the migration period, it still reduced cell migration 20%, and this deficit could not be restored by concomitant addition of the polyamines. Intracellular polyamine levels at these times, i.e., 6 h or 4 days, were an important factor in these results. Only PUT was undetectable after 6 h of DFMO. SPD and SPM were still at normal levels at 6 h. SPD was undetectable at 4 days, but SPM was still at 40% of normal. These data give added importance to PUT because its absence reduced cell migration after only 6 h, while SPD and SPM were still present in normal amounts. Perhaps exogenous SPD and SPM restored cell migration when present with DFMO for 4 days treatment primarily because they contributed to intracellular PUT through the acetyltransferases.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Decreased expression of protooncogenes c-fos, c-myc, and c-jun following polyamine depletion in IEC-6 cells.

Direct exposure of small intestinal mucosal cells to luminal polyamines stimulates proliferation. This study tests the hypothesis that the protooncogenes c-fos, c-myc, c-jun, and junB are involved in the mechanism by which polyamines modulate mucosal growth. Studies were conducted in the IEC-6 cell line, derived from rat small intestinal crypt cells. Cells were grown in Dulbecco's minimal essential medium containing 5% dialyzed fetal bovine serum (dFBS) in the presence of absence of alpha-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase, which is the rate-limiting enzyme for polyamine synthesis. Cellular polyamine levels, cell growth, and relative abundance of c-fos, c-myc, c-jun, and junB mRNAs, were measured at 1, 2, 4, 6, 8, and 12 days after initial plating. The intracellular polyamines, spermidine and spermine, and their precursor, putrescine, in DFMO-treated cells decreased significantly at 2 days and remained depleted thereafter. Although DFMO profoundly decreased growth and final cell number, both control and DFMO-treated cells entered a plateau phase by 6 days. In control cells, c-myc and c-jun mRNA levels significantly increased on days 4-6 and then returned to a basal level of expression, which was maintained thereafter. c-fos mRNA in quiescent cells after 24 h serum deprivation was significantly stimulated by 5% dFBS, although a steady-state level of c-fos mRNA was undetectable in control cells. Treatment with DFMO not only prevented increased expression of c-myc and c-jun protooncogenes at 4 days, but also significantly reduced steady-state levels of c-myc and c-jun mRNA between 6 and 12 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Migration of IEC-6 cells: a model for mucosal healing.

Cell migration is the principal force behind the early restitution of erosions of the mucosa of the gastrointestinal tract. Despite the importance of cell migration to healing, no attempts to study the process in culture have been reported. We have attempted to standardize conditions for migration and test the migration responses of the small intestinal epithelial crypt cell line IEC-6 in some experimental situations already well known in vivo. We found good correspondence between in culture and in vivo on the following points: 1) migration was independent of DNA synthesis; 2) DNA synthesis was not concentrated at the wound edge; and 3) inhibition of actin polymerization stopped migration altogether. In addition, the presence of an extracellular matrix maximized migration. Protein inhibitors with different modes of action inhibited cell migration to different degrees, not always commensurate with their inhibition of protein synthesis. Cell surface proteoglycans were important; hyaluronic acid had an effect, but the secretion of a migration-stimulating substance by wounded cells was equivocal. Significantly, alpha-difluoromethylornithine (DFMO), which inhibits ornithine decarboxylase and polyamine synthesis, almost totally prevented cell migration. Because DFMO also prevents healing of mucosal erosions in vivo, we believe that this model can be used, keeping in mind its spatial limitations, to study the process of cell migration involved in the early restitution of mucosal erosions.

Cell Communication↗
PubMed1992

Effect of putrescine on S-adenosylmethionine decarboxylase in a small intestinal crypt cell line.

Two key enzymes in polyamine biosynthesis are ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC). SAMDC decarboxylates S-adenosylmethionine, which then donates aminopropyl groups for spermidine and spermine synthesis. The purpose of our study was to determine whether putrescine, taken up from medium or synthesized endogenously by ODC, alters SAMDC activity. Studies were conducted in the IEC-6 cell line derived from rat small intestinal crypt cells. Cells were grown in Dulbecco's minimal essential medium containing 5% dialyzed fetal bovine serum (dFBS). They were deprived of serum for 24 h before experiments. Basal SAMDC activity was increased significantly by > or = 10(-4) M of putrescine. Lower doses had no significant effect. The same doses of putrescine decreased ODC activity to near zero. Asparagine at 10 mM or 5% dFBS not only stimulated ODC activity and the intracellular putrescine levels but also increased significantly SAMDC activity as well. ODC activity peaked at 3 h, and the maximum level of SAMDC occurred 3-4 h after exposure to asparagine or serum. Treatment with DL-alpha-difluoromethylornithine (DFMO), a specific ODC inhibitor, prevented the increases in both cellular putrescine levels and SAMDC activity in asparagine- and serum-treated cells. In the presence of DFMO, exogenous putrescine returned SAMDC activity toward control levels but had no effect on ODC. A very slight increase of SAMDC half-life in IEC-6 cells grown in the presence of putrescine was not statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosylmethionine Decarboxylase↗
PubMed1991

Putrescine uptake and release by a normal rat small intestine crypt cell line, IEC-6.

IEC-6 cells were cultured on permeable filter inserts with separate access to the apical and basolateral sides. [3H]Putrescine uptake favored the apical side and its release (in Earle's balanced salt solution containing 0.1% bovine serum albumin) was six times greater in the apical-to-basolateral than in the basolateral-to-apical direction. Release in DMEM did not show this preference. The uptake of [3H]putrescine was stimulated approximately 1.3 times the basal level by 10 mM asparagine (ASN) or 5% dialyzed fetal bovine serum whether the [3H]putrescine was added at a concentration of 1 or 100 nM. The increased uptake was maintained for up to 6 h. When [3H]putrescine was removed after 4 h of uptake, the cells continued to release it into the medium on both sides for up to 4 h. Stimulated cells released only 50% as much as unstimulated cells. Unlabeled putrescine reduced the uptake of [3H]putrescine with an IC50 of 1.81 x 10(-6) M (r = 0.9476) and 1.02 x 10(-6) M (r = 0.9967) for unstimulated and ASN-stimulated cells, respectively. When the intracellular putrescine was reduced by difluoromethylornithine, the uptake of [3H]-putrescine was not changed, but its release was inhibited. Sodium was not required for [3H]putrescine uptake or release. Although the stimulated cells attained intracellular levels of [3H]putrescine which, if expressed as concentration based on cell volume, were up to 500 times the original extracellular concentration, a true concentration gradient could not be proven because 85% of the [3H]putrescine was probably bound to polyanions as shown by butanol extraction.

Animals↗
PubMed1991

Role of polyamines in gastrointestinal mucosal growth.

The polyamines have been under active investigation for nearly three decades. There is a great deal of evidence that they play an important role in gastrointestinal mucosal growth, but the mechanisms through which this role is carried out are still not fully explained. This review examines the role of the polyamines in the regulation of mucosal growth, the control of intracellular polyamine levels, the biosynthesis of the polyamines, and some known mechanisms of their action. Finally, we propose a model of polyamine action that reconciles the effects of various trophic agents and situations in which growth is stimulated along with concomitant changes in polyamine levels. It accounts for both humoral and gradient-oriented features of various adaptive responses of the gastrointestinal mucosa and is intended to provide a framework for the future investigation of the role of the polyamines in this tissue.

Animals↗
PubMed1991

Stimulation of proximal small intestinal mucosal growth by luminal polyamines.

The purpose of this study was to determine whether luminal polyamines stimulate intestinal mucosal growth in vivo. Rats received 2% alpha-difluoromethylornithine (DFMO) added to their drinking water throughout the experiment. The polyamines spermidine and spermine (3 mg each/100 g body wt) were given intragastrically in combined doses once at 9:30 A.M. and again at 5:30 P.M. Duodenal and jejunal mucosal ornithine decarboxylase (ODC) activity in the DFMO-treated rats was inhibited significantly for the duration of the study. DFMO also markedly decreased the rate of [3H]thymidine incorporation into DNA of duodenal and jejunal mucosa. The decrease in [3H]thymidine incorporation was significant 4 days and maximal 6 and 8 days after beginning treatment with DFMO. Decreased ODC activity and DNA synthesis were paralleled by decreases in total mucosal DNA, RNA, and protein content. Administration of the polyamines significantly reversed the effects of DFMO except the inhibition of ODC. In fact, there were no significant differences in mucosal growth parameters between the controls (without DFMO) and those treated with DFMO plus polyamines. Oral administration of spermidine and spermine at a dose of 4.5 mg each/100 g body wt for 6 days to rats not treated with DFMO increased the normal rate of mucosal growth in the duodenum and jejunum as well. Polyamine accumulation in IEC-6 cells was measured to determine whether it was altered by DFMO. IEC-6 cells took up [3H]putrescine and [3H]spermidine from their surrounding environment and the uptake was stimulated by serum. DFMO (5 mM) totally inhibited the increase in ODC activity but had no effect on the cellular uptake of polyamines in the presence of putrescine.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Regulation of ornithine decarboxylase activity in LoVo cells.

The role of Na+ and Na(+)-H+ exchange in the stimulation of ornithine decarboxylase (ODC) activity has been investigated in a human colon adenocarcinoma cell line, LoVo. Asparagine (Asn; 10 mM) or 10% fetal bovine serum (FBS) increased ODC activity from undetectable levels to greater than 500 pmol CO2.mg protein-1.h-1 in 4 h. This increase could be reduced 50% by concentrations of Na(+)-H+ exchange inhibitors that did not reduce protein synthesis. (approximately 0.2 mM for amiloride and 0.05 mM for hexamethyleneamiloride). Asn was able to double the uptake of 22Na+, whether an ionic (choline chloride) or nonionic (D-mannitol) substance was substituted for Na+, and the substitution of these compounds as well as N-methyl-glucamine for Na+ largely prevented the stimulation of ODC by Asn. Another factor influencing ODC activity was extracellular pH (pHo). When pHo was lowered, intracellular pH (pHi) also fell, and ODC activity was reduced. When pHo was raised, pHi also rose, and ODC activity increased. The well-known correlation between increased pHi and Na+ uptake with the stimulation of growth may be due to their influence on ODC activity.

Adenocarcinoma↗
PubMed1989

Putrescine uptake and release by colon cancer cells.

We have investigated the uptake and release of [3H]putrescine by a human colon adenocarcinoma cell line (LoVo) maintained on filter inserts. This culture system permits the cells to develop morphological polarity and provides separate access to the basolateral and apical surfaces of the cells. [3H]putrescine was taken up more readily by the basolateral than by the apical side of the cells. [3H]putrescine uptake could be stimulated greater than 300 times by either 10 mM asparagine or 10% fetal bovine serum. [3H]putrescine was accumulated to a concentration gradient of approximately 300-fold; uptake could be inhibited 50% by 7.5 microM unlabeled putrescine and was not dependent on Na+. The release of [3H]putrescine into the apical medium was inhibited by asparagine or fetal bovine serum. Usually, less than one-thousandth of the [3H]putrescine taken up into the cells was released into the apical medium. Release of [3H]putrescine did not correspond to the accumulation of [14C]-inulin in the apical medium. For these reasons we concluded that putrescine release was not simply passive leakage but was responsive to intracellular demand. The [3H]putrescine taken up by the cells as well as that released into the apical medium was greater than 90% unmetabolized at 4h.

Amiloride↗
PubMed1984

Methodological aspects of analysing human breast cancer cell lines by NMR spectroscopy.

In an attempt to identify the factors which might affect the measurement of water proton relaxation times in cultured cells, we have begun a long-term study of two human breast cancer cell lines, MDA-MB-231 and MDA-MB-435s. We tested growth rates and cell cycle distribution as intrinsic properties of the cells as well as methodological steps which might affect the measurement of T1 and T2. A detailed examination of the growth rates of the two cell lines, easily recognized as slow (231) and fast (435s) in culture, revealed that this attribute is difficult to correlate precisely with T1s or T2s. The reason is that the relaxation times are necessarily measured at one point in time while the growth rates are a summation of ongoing processes occurring over hours. Cell cycle distribution, on the other hand, can be measured simultaneously with the relaxation times by using cells quick-frozen from the same suspension. By this method, cell cycle distribution appears to be reflected through an effect on T1s. For example, cell pellets distributed 72:15:14 in G0G1:S:G2M has longer T1s (p less than 0.01) than those distributed 43:34:23 in G0G1:S:G2M. Regarding methodological factors, trypsin appeared to lower water content and T2s in the 231 cell line. Drift in the cell cycle distribution after sample preparation did not become significant until after 2 hours in the NMR tube. It was important to standardize the force and duration of centrifugation of the cell pellets to minimize the contribution of the suspending medium without affecting cell viability. We conclude that, given careful control of methodological factors, differences in T1 may reflect metabolic differences as demonstrated by T1 differences in cell pellets showing divergent cell cycle distribution.

Breast Neoplasms↗
PubMed1980

Differential response of individual uterine cell types from immature rats treated with estradiol.

The separation of viable epithelial, stromal, and myometrial cells from immature rat uteri is described. The integrity of the separated cells was established by their ability to grow in culture and to exclude trypan blue and by transmission and scanning microscopy. Epithelial preparations contain insignificant contamination by other cells. Stromal fractions are estimated to contain 10% epithelial contamination. Myometrial fractions probably contain significant contamination by stroma, but the amount is difficult to quantify. The three cell types separated from untreated rats or from rats treated with 17 beta-estradiol (E2) differ from each other physiologically. Control epithelial cells incorporated more tritiated cytidine per microgram DNA than stromal or myometrial cells. 17 beta-Estradiol enhanced incorporation of tritiated cytidine into RNA of epithelial cells but not stromal or myometrial cells. RNA to DNA ratios of cells from untreated rats were 1.0 (epithelial), 0.5 (stromal), and 0.6 (myometrial). RNA to DNA ratios rose approximately 30% in all cell types after E2. Estrogen receptor (ER) concentrations, determined by Scatchard analysis under exchange conditions, also differed in the three cell types. Nuclear ER concentrations (as measured in crude nuclear fractions) were significantly higher (P less than 0.01) in epithelial and stromal cell fractions from untreated animals than in the myometrial fraction, i.e. 5.5, 3.8, and 1.2 fmol/microgram DNA, respectively. The chief effect of E2 administration was a significant increase (P less than 0.05) in cytosol ER of all cell types. Epithelial cells had the highest cytosol ER concentration, increasing from 0.5 to 3.1 fmol/microgram DNA 48 h after E2 administration. Although the myometrial cells had the lowest ER concentration, they contained 84-89% of the entire uterine ER by virtue of the proponderance of myometrium in the whole uterus. The differing responses of epithelial and stromal cells, therefore, are diluted or masked by the ER content of the myometrium in assays of whole uterus. Although the total number of ER sites in the cytosol and nucleus (16,900-32,100 sites/cell) in separated stromal cells at various times after E2 stimulation was similar to ER concentrations (20,000-30,000 sites/cell) previously reported for the whole uterus, the partition of ER between cytosol and nucleus (10:90) was different, favoring the nucleus even before E2 administration. ER began to appear in increased amounts in the cytosol only 24 h post E2 administration. When tritiated E2 was given in vivo and its specific binding was assessed in the uterine cells subsequently separated, ER partition between cytosol and nucleus was also heavily in favor of the nucleus. However, when whole uterus was assayed, the partition of ER between cytosol and nucleus (90:10) was exactly the opposite. Possible reasons for this unusual finding are discussed.

Animals↗