Chronic inflammation and cancer: potential role of Bcl-2 gene family members as regulators of cellular antioxidant status.

The incidence of cancer is increased in all gastrointestinal (GI) tissues as a result of chronic inflammation. These cancers may develop in cells that are selected for resistance to inflammatory products by virtue of overexpressing the antioxidant protein Bcl-2 or other Bcl-2 gene family members that have antioxidant activity. Unfortunately, Bcl-2 also functions as an anti-apoptotic. Bcl-2 overexpression can increase cellular lifetime and increase the likelihood that other cancer-related mutations will accumulate in individual cells. Thus, Bcl-2 expression is cytoprotective, but its expression also increases the risk of cancer incidence. This is perhaps more evident in the GI tract, which is exposed to more potential mutagens relative to other tissues.

Immunohistochemical evaluation of Bcl-2 gene family expression in liver of hepatitis C and cirrhotic patients: a novel mechanism to explain the high incidence of hepatocarcinoma in cirrhotics.

OBJECTIVE: The purpose of this study was to determine whether there is an increase in expression of bcl-2 and related bcl-2 gene family members bcl-X and bax in liver biopsy samples obtained from patients with either hepatitis C infection or cirrhosis. Bcl-2, bcl-X, and bax, as well as other bcl-2-related proteins, function coordinately through homo- and heterodimerization to regulate apoptosis. Bcl-2, which is characterized as an antiapoptotic, also functions as an antioxidant. We hypothesized that a mechanism that could account for increased hepatocellular carcinoma in patients with hepatitis C and cirrhosis is selection of bcl-2 expressing cells. This selection would be due to the capacity of individual cells to resist the toxic effects of inflammatory byproducts, specifically reactive oxygen species. METHODS: Sections cut from archived liver biopsy samples embedded in paraffin were probed with antibody specific for bcl-2, bcl-X, or bax. Liver samples were from normal (N = 5), hepatitis C patients (N = 19), and cirrhotics (N = 10). Percent positive staining and intensity of staining were judged independently for hepatocytes, bile ducts, mononuclear cells, and Kupffer cells. RESULTS: Bcl-2 expression was evident in bile ducts and mononuclear cells of hepatitis C patients, but was not commonly present in hepatocytes (two of 10). In the cirrhotic liver, bcl-2 expression was also detected in bile ducts and mononuclear cells, but in contrast to hepatitis patients was also expressed in hepatocytes (nine of 10). A similar pattern of expression was evident for bcl-X, but in general the level of expression was limited relative to that of bcl-2. Bax expression was infrequently present in sections from any of the three patient groups. CONCLUSIONS: The data indicate that bcl-2 expression is elevated in the liver of cirrhotics, but is not evident in the liver of hepatitis C patients. This increase in expression of bcl-2 in cirrhotic patients may correlate with development of hepatocellular carcinoma given the anti-apoptotic/oncogenic potential of bcl-2.

Determination of protein carbonyl groups by immunoblotting.

Free radical-mediated oxidation of proteins results in the formation of carbonyl groups in quantities that reflect the intensity of the oxidative stress. We have developed an immunochemical technique for the quantification of carbonyl groups in protein samples prepared from small tissue samples and cell cultures. Protein samples were slot-blotted onto a polyvinylidene difluoride membrane, which was sequentially treated with 2,4-dinitrophenylhydrazine (DNPH), a primary antibody specific for the 2,4-dinitrophenol group, and a peroxidase-labeled second antibody. After the blots were developed with a chemiluminescent substrate and exposed to X-ray film, the level of immunostaining was quantitated by densitometry. Using oxidized bovine serum albumin as a standard and loading 5 microg of protein per slot, the minimum detectable carbonyl content was approximately 60 pmol carbonyl/mg protein. When necessary, nonspecific staining by noncarbonyl constituents in complex sample matrices was accounted for by using sodium borohydride-treated blanks. Results by the new method were highly correlated (r = 0.932, P < 0.0001) with those of the standard DNPH-based spectrophotometric technique. The coefficient of variation at a carbonyl level of 1.5 nmol/mg protein was 9.7%. The utility of this new method was demonstrated by measuring protein oxidation in cultured human colon cells (SW620) that were briefly exposed to H2O2.

Beneficial effect of a bile acid resin binder on enteral feeding induced diarrhea.

OBJECTIVES: Diarrhea is a complication of enteral feeding, occurring in up to 68% of critically ill patients. We hypothesized that prolonged fasting results in abnormal bile acid homeostasis. Subsequent enteral feeding then causes a relative luminal excess of bile acids, which leads to choleretic diarrhea. Hence, diarrhea induced by enteral feeding should improve with the use of a bile acid binding agent, such as Colestid Granules. METHODS: We evaluated the effect of Colestid on enteral feeding-induced diarrhea in a double-blind placebo-controlled study. Nineteen patients who were nil per os (NPO) for 5 days before initiation of enteral feeding were enrolled in the study and treatment continued for 7 days. The severity and frequency of diarrhea were quantified. Fecal bile acids were measured enzymatically. Stool nutrient loss was measured by fat extraction, microkjeldahl determination of nitrogen, and bomb calorimetry of dried fecal specimens. RESULTS: Enteral feeding resulted in a high frequency of diarrhea (95%) at some time during the observation period. The majority of episodes of diarrhea in both groups were of low volume. Colestid significantly decreased the prevalence and severity of diarrhea. Colestid had no significant effect on fecal calorie or nutrient losses. The average bile acid concentration in the stool increased significantly after enteral feeding. CONCLUSION: Enteral feeding-induced diarrhea is, at least in part, due to malabsorption of bile acids. The bile acid resin binding agent Colestid improves diarrhea induced by enteral feeding.

Aspirin toxicity for human colonic tumor cells results from necrosis and is accompanied by cell cycle arrest.

Chemoprevention of colorectal cancer using aspirin has been demonstrated in rodents and has been suggested by data from epidemiological studies. The mechanism that accounts for this prevention is unknown, but it is thought to relate to an irreversible inhibition of cyclooxygenase and, subsequently, prostaglandin production. The effect of aspirin on the growth of human colonic tumor cells was determined in an effort to gain insight into a possible mechanism of action. In the two cell lines studied, SW 620 and HT-29, we observed a significant dose- and time-dependent increase in aspirin toxicity in a concentration range of 1.25-10 mM. This result was independent of prostaglandin production, because there was no measurable prostaglandin E2 in cell culture medium. As compared with controls, cells in cultures that contained aspirin were not detached, which suggests that the mechanism of cell death was not apoptosis. Flow cytometric analysis revealed an increase in S phase and G2-M populations as well as the number of subdiploid nuclei in cultures treated with high-dose aspirin. Confirmation that cells were undergoing necrosis in response to aspirin was evident from the presence of cells that bound annexin V and accumulated propidium iodide in the absence of a population that bound annexin alone. The results suggest that aspirin induces cell cycle arrest and causes necrosis at high concentrations in vitro, but does not induce apoptosis. Collectively, these two events, necrosis and cell cycle arrest, may contribute to the chemopreventive effect that seems to result from long-term administration of aspirin in vivo.

CD11b mRNA expression in neutrophils isolated from peripheral blood and gingival crevicular fluid.

Adhesion molecule CD11b/CD18 expressed by neutrophils (PMNs) participates in cell migration and phagocytosis of C3bi derivatized bacteria. It is this phagocytic function that eliminates some of the known periodontal pathogens in periodontal pockets. In patients with advanced periodontitis, homotypic aggregation of crevicular fluid PMNs (CF-PMNs) may occur due to overexpression of CD11b/CD18 and this may lead to ineffective elimination of periodontal pathogens. We have previously shown that CF-PMNs isolated from the periodontal pockets overexpress CD11b compared to PB-PMNs. This study tested the hypotheses that (1) overexpression of surface CD11b correlates with expression of CD11b mRNA in CF-PMNs isolated from advanced periodontitis subjects, and (2) the intrinsic capacity of CD11b mRNA upregulation by PB-PMNs from periodontitis patients differs from that of control subjects. CF-PMNs and peripheral blood PMNs (PB-PMNs) were isolated from 13 subjects with healthy gingiva (control group) and 13 subjects with advanced periodontitis (patient group). The surface expression of CD11b was determined by flow cytometry and CD11b mRNA was determined by extraction of mRNA and reverse transcription to cDNA followed by DNA amplification using primers to detect a segment of the cDNA which encodes CD11b. The results of this study confirm that the surface expression of CD11b on CF-PMNs is significantly higher in periodontitis subjects vs control subjects (p = 0.03), whereas surface CD11b expression on PB-PMNs does not differ significantly between groups (p = 0.06). The level of surface CD11b expression on CF-PMNs did not correlate with the amount of mRNA present in CF-PMNs in either group (p = 0.056, 0.07 for control and periodontitis patients, respectively). Most (9 of 13) individuals in the patient group expressed CD11b mRNA whereas very few control subjects (2 of 11) had CD11b mRNA in their CF-PMNs. This difference between groups was statistically significant (p = 0.004). The capacity to upregulate CD11b mRNA upon stimulation with fMLP and/or GM-CSF was highly variable and there was no statistical difference between the 2 groups.

Suramin is synergistic with vinblastine in human colonic tumor cell lines: effect of cell density and timing of drug delivery.

Suramin is a multi-targeted antiproliferative drug developed for the treatment of African trypanosomiasis but with potential efficacy for the treatment of human cancer. Cell growth inhibition was determined in vitro for three human colonic tumor cell lines using three different doses of suramin (50, 100 and 200 microM). At the lower suramin concentration cell growth was stimulated relative to control cultures in all three cell lines. At the higher dose which is at the upper end of the tolerable dose in humans suramin reduced cell numbers by greater than 50%. Inhibition of cellular proliferation was reduced relative to increases in cell plating density. Addition of vinblastine six and to a lesser extent 72 hours post suramin (200 microM) resulted in an inhibition of cell growth and/or toxicity that exceeded that which occurred as a result of exposure to either suramin or vinblastine alone. To investigate the possible mechanism by which suramin sensitizes cells to vinblastine we determined the effect of suramin on expression of the multidrug resistance (mdr1) gene. A decrease in mdr1 mRNA was evident in one colon tumor cell line and a slight decrease detected in a second line. The results establish that suramin is effective in controlling growth in colonic tumor cells and confirms that suramin activity is synergistic with other chemotherapeutics. The effect of suramin on MDR is of potential value that needs to be more thoroughly investigated particularly in cancers such as the those of the colon that are often drug refractory.

Effect of aspirin on prostaglandin E2 and leukotriene B4 production in human colonic mucosa from cancer patients.

Results from epidemiological studies indicate that chronic administration of aspirin reduces the incidence of colon cancer. The mechanism that accounts for this reduction is not known, but it may be related to the decreased production of prostanoids that results from aspirin inhibition of cyclooxygenase. However, it is not known whether aspirin has a local effect on prostanoid production in the colonic mucosa and whether this effect is dose dependent. In this study, we determined the effect of oral administration of aspirin on the production of the prostanoid prostaglandin E2 (PGE2) in the intact human colonic mucosa. Inhibition of cyclooxygenase could result in an increased availability of arachidonic acid and a corresponding increase in production of other eicosanoids. To determine whether such an effect occurs, we also quantitated the concentration of leukotriene B4 (LTB4) in colonic mucosal samples. Mucosal samples were obtained during sigmoidoscopy from the colons of 17 subjects with a history of colonic cancer prior to and following 60 days of self-administration of 325 mg aspirin/day and again 60 days after administration of 650 mg aspirin/day. PGE2 and LTB4 concentrations were determined by enzyme immunoassay for tissue samples that were flash frozen after removal from the biopsy forceps and also in medium that was collected from tissue samples that were incubated for 4 h following removal from the subject. PGE2 concentrations were decreased significantly in samples collected after 60 days of consumption of 325 mg aspirin. An additional 60 days of consuming 650 mg aspirin/day did not result in a further significant decrease relative to that attained after consumption of 325 mg/day. Similar results were obtained using colonic explants, and the addition of aspirin to medium further reduced PGE2 production. LTB4 in tissue and medium was not significantly different in pre-versus post-aspirin samples, with the exception of an increased concentration in medium samples collected after consumption of 650 mg/day relative to pre-aspirin samples. The results indicate that aspirin affects eicosanoid production in the colonic mucosa of humans, but the effect is most likely restricted to products of the cyclooxygenase-dependent pathway. It appears that 325 mg of aspirin is sufficient to affect PGE2 production and that increasing the dosage to 650 mg daily provides an additional decrease in PGE2 synthesis. However, the higher dosage was associated with a considerable increase in complaints of gastric discomfort. Additional study is needed to establish whether doses less than 325 mg also provide a significant decrease in PGE2 production.

Expression of c-myc in human colonic tissue in response to beta-carotene supplementation.

Dietary supplementation with beta-carotene at 30 mg/day results in an increased serum trans-retinoic acid concentration in patients with a prior colonic polyp. In a number of human cell lines, trans-retinoic acid upregulates c-myc mRNA expression in colonic mucosa by reverse transcription polymerase chain reaction and correlated the results with serum concentrations of all-trans- (ATRA), 13-cis-(13-cRA), and total retinoic acid. Serum and colonic biopsy samples were obtained before and 90 days after administration of a placebo (n = 7) or 30 mg of beta-carotene (n = 5) daily. An increase in c-myc expression after supplementation was observed in 6 of 12 subjects, but 5 of these 6 subjects had decreased total serum retinoic acid concentration and 4 had decreased ATRA concentration. In addition, five of the six subjects with increased c-myc expression had received a placebo. Conversely, c-myc expression was increased in only two of five paired samples from subjects whose total serum retinoic acid concentration increased during the 90-day supplementation period. We conclude that c-myc expression is not correlated with ATRA, 13-cRA, or total retinoic acid concentration in vivo and that increased serum retinoic acid secondary to increased tissue beta-carotene is not sufficient to activate c-myc transcription.

Immunomodulatory effect of beta-carotene on T lymphocyte subsets in patients with resected colonic polyps and cancer.

Results from a number of studies suggest that beta-carotene-containing foods prevent the initiation or progression of various cancers. One possible mechanism for this effect could be enhancement of the immune response. The aim of this study was to determine whether beta-carotene modulates T lymphocyte subsets in patients affected with colonic polyps or cancerous lesions. Patients with previous adenomatous colonic polyps (n = 18) or colon cancers (n = 19) were randomized to receive placebo or beta-carotene (30 mg/day) for three months. Percentages of T lymphocyte subsets were determined using flow cytometry in blood samples collected before randomization and at three months. T lymphocyte subsets of 14 normal control subjects were also determined for comparison. Initially, there was no difference in total leukocyte counts, percentage of lymphocytes, and various subsets of lymphocytes among the three groups, although in cancer patients there was a lower percentage of CD4 and interleukin-2 (IL-2) receptor-positive (IL-2R+) cells than in patients with polyps and in controls. After supplementation with beta-carotene, a significant increase in IL-2R+ T lymphocytes (from 12.7 +/- 3.0% to 26.0 +/- 1.9%) and CD4+ lymphocytes (from 40.9 +/- 3.1% to 45.6 +/- 3.2%) was seen only in the cancer patients. These percentages remained unchanged in patients with adenomatous polyps receiving placebo or beta-carotene. We concluded that beta-carotene increased the number of IL-2R+ T lymphocytes and CD4+ lymphocytes, which in turn may produce IL-2 only in patients with cancer who may already have some deficiency in their immune system. This increase in activated T lymphocytes may mediate cytotoxic reactions to cancer cells via cytokine production.

Cytotoxic effect of beta-carotene in vitro is dependent on serum concentration and source.

In a previous study we reported that beta-carotene solubilized in tetrahydrofuran (THF) is toxic for human colonic tumor cells in vitro using media containing 10% fetal calf serum (FCS). Cytotoxicity was evident using beta-carotene concentrations that are achieved in human serum as a result of supplementation with 30 mg beta-carotene/day. In an attempt to determine the mechanism for this toxicity we investigated the effect of beta-carotene when present in human serum as a result of dietary supplementation. This effect was compared to that observed for cells incubated in THF-solubilized beta-carotene. The results indicate that human serum from subjects with a high concentration of beta-carotene is not cytotoxic. Subsequent analysis revealed that in contrast to the results using FCS, THF-solubilized beta-carotene is not cytotoxic in the presence of human serum. In addition, the effect observed with FCS is blunted with increasing FCS concentration from > or = 90% cytotoxicity using 10% FCS to 36% using 100% FCS. The difference in results obtained using FCS and human serum may be due to a serum component that is relatively lacking in FCS as compared to human serum.

Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans and Treponema denticola detection in oral plaque samples using the polymerase chain reaction.

Detection of putative pathogens is critical for delineating the etiology and progression of periodontitis. In the present study, we have used a polymerase chain reaction (PCR) assay utilizing primers specific for the lkt A gene of Actinobacillus actinomycetemcomitans, the fimbrial gene of Porphyromonas gingivalis, and tdp A gene of Treponema denticola in order to determine the presence of these pathogens in subgingival plaque samples from periodontitis sites. These gene specific primers were also used to assess the detection of different strains of bacteria in the PCR assays. Primers for P. gingivalis detected P. gingivalis strain 33277, but no product was detected when primers were used with extracts from 4 species of Capnocytophaga, 3 species of Prevotella, 2 species of Porphyromonas other than P. gingivalis, Bacteroides levii, Escherichia coli, 3 strains of A. actinomycetemcomitans and 3 strains of T. denticola. PCR analysis using primers for the lkt A gene of A. actinomycetemcomitans also did not result in a product with any of these bacteria with the exception of a positive result with 3 different strains of A. actinomycetemcomitans. Primers selected from the tdp A gene of T. denticola did not identify any of the bacteria strains tested except T. denticola serovars a, b, and c. Thus, these primers were shown to amplify gene segments that are specific to either P. gingivalis (33277), A. actinomycetemcomitans (33384, 43717 and 43718) or T. denticola (35405, 33521 and 35404). The PCR assay may be used to rapidly detect the presence of periodontal pathogens in the future.

In vitro beta-carotene toxicity for human colon cancer cells.

Experiments were conducted to determine the effect of beta-carotene on human colon cancer cells in vitro. beta-Carotene solubilized in tetrahydrofuran (THF) was determined to be cytotoxic for three different cell lines: LS 180, SW 620, and HCT-15. The number of LS 180 and SW 620 cells surviving treatment with 2.9 microM beta-carotene was significantly reduced relative to THF-treated cells, and a similar reduction was achieved in HCT-15 cells with use of 5.8 microM beta-carotene. These concentrations are in the range achieved in serum of individuals supplemented with beta-carotene at 30 mg/day. There was no beta-carotene cytotoxicity in the concentration range that characterizes serum of unsupplemented individuals. Vitamin E at > 200 microM was not cytotoxic and at higher concentrations slightly stimulated proliferation of all three cell lines. Exposure of cells to vitamin E did not diminish the cytotoxicity of beta-carotene, suggesting that the toxic effect of beta-carotene is not due to prooxidant activity. Percent cytotoxicity was increased by extending the duration of exposure of cells to beta-carotene. Interestingly, beta-carotene cytotoxicity decreased with increasing cell density. This density-dependent toxicity was attributable to a higher beta-carotene concentration per cell for cells plated at lower densities. Thus toxicity of beta-carotene for colon cancer cells is dose, time, and cell density dependent and occurs in vitro at concentrations that can be achieved safely in humans.

Effect of beta-carotene supplementation on indices of colonic cell proliferation.

BACKGROUND: Epidemiologic studies have shown that consuming foods containing beta-carotene is associated with a decreased incidence of colon cancer. The validity of this association has recently been questioned. It is not known if the rate of colonic cell proliferation differs among individuals with or without a history of colonic polyps or cancer and if proliferation changes in response to beta-carotene. PURPOSE: This study was intended to (a) determine whether differences exist in colonic cell proliferation in individuals with and without prior colonic polyps or tumors, (b) demonstrate that beta-carotene accumulates in colonic mucosa following dietary supplementation, and (c) determine whether mucosal beta-carotene accumulation influences colonic cell proliferation. METHODS: Subjects were enrolled in the phase I study from June 1991 until February 1994. The participants included 20 individuals (11 males and nine females, aged 62.3 +/- 8.9 years [means +/- SD]) with normal colons (as judged by recent colonoscopy), 40 (24 males and 16 females, aged 59.6 +/- 10.1 years) with a history of colonic polyp(s), and 41 (30 males and 11 females, aged 67.2 +/- 9.7 years) with prior colon cancer. The subjects in the last two groups consumed either 30 mg of beta-carotene or placebo each morning for 3 months. This dose of beta-carotene has no known toxic effects, but it can increase the serum level by approximately 10-fold. beta-carotene concentration in serum and colonic tissue was quantitated by high-pressure liquid chromatography in samples collected before and after supplementation with beta-carotene or placebo. Cellular proliferation was assessed on the basis of tissue ornithine decarboxylase activity, urinary polyamine excretion, and proliferating cell nuclear antigen expression. The differences in colonic cell proliferation parameters due to beta-carotene supplementation, within and among different groups, were evaluated by the Wilcoxon matched-pairs signed ranked test and the Mann-Whitney test, respectively. All statistical tests were two-sided. RESULTS: Colonic cell proliferation did not differ in samples obtained from individuals with and without prior colonic polyp(s) or cancer. beta-carotene concentrations in serum and colonic tissue were significantly increased in groups receiving beta-carotene (P < .001). However, cell proliferation did not differ, as judged by any of the three measures, among samples from all experimental groups collected before and after supplementation with beta-carotene. CONCLUSIONS: Dietary supplementation with beta-carotene for a period of 3 months does not alter colonic cell proliferation in individuals with a history of colonic polyps or cancer. IMPLICATIONS: The mechanism by which beta-carotene might reduce colon cancer incidence does not appear to involve or result in a change in cell proliferation in the normal colonic mucosa as studied in individuals with a history of colonic polyps or cancer.

P-glycoprotein expression by human colonic tumor cells influences the growth of tumor cells in vitro.

The stability of P-glycoprotein expression by high and low expressing cells obtained by flow sorting from two cell lines established from colonic tumors was assessed. Low expressing cells rapidly recovered expression to a level that was comparable to that determined for uncloned cells. High expressing cells maintained a high level of expression relative to the parental uncloned lines, although there was continuous reversion to a level of expression determined for the parental line with time. The population growth rates for high expressing cells was greater than that of parental and low expressing cells when assessed immediately following plating of sorted cells. The data suggest that high expression of P-glycoprotein confers a growth advantage which may increase repopulation and dissemination of tumor cells following drug therapy.

Accumulation of beta-carotene in normal colorectal mucosa and colonic neoplastic lesions in humans.

The quantity of beta-carotene (BC) accumulated in colonic polyps and colonic cancerous tissue in humans in situ was determined relative to the quantity accumulated in normal colon and rectal tissue. Serum concentration of BC, retinol, and alpha-tocopherol and tissue BC concentration were determined by high-performance liquid chromatography in samples obtained before and after oral supplementation with BC (30 mg/day). The serum BC and retinol concentrations significantly increased in response to supplementation in control, polyp, and cancer patients, but there was no change in serum alpha-tocopherol concentration. The BC concentration in tissue (colon, rectum, and tumor) of cancer patients was significantly less than that in tissue samples from control and polyp patients. Relative to baseline values, BC accumulated to a significant extent in tissues from all patients, including polyp and tumor tissue, during supplementation. The results indicate that BC does accumulate in colonic neoplastic tissue in humans and may potentially be utilized to augment cytotoxicity of chemotherapeutics or to prevent malignant transformation of cells.

Assessment of the intestinal retention of beta-carotene in humans.

OBJECTIVE: Assessment of the intestinal absorption of beta-carotene (BC) in humans as well as plasma clearance of BC has been difficult. We have used the total gut washout method (TGWM) to assess BC retention during transit through the intestine, as well as the effect that different diets and age have on BC retention. METHODS: HPLC was used to quantitate fecal and serum BC concentrations from young and elderly subjects who had undergone the TGWM to remove all intestinal contents prior to ingesting BC or placebo with or without a meal. Meals contained different combinations of calories and fat. RESULTS: In subjects receiving no meal, 83% of ingested BC was recovered in rectal effluent collected within 24 hours post-BC administration. The quantity of BC in feces of individuals receiving meals was 49-71%. There was no significant change in serum concentrations of other carotenoids or retinoids following consumption of BC with any of the different meals. Interestingly, both diet and age influenced the efficiency of BC absorption. An increase in dietary fat content resulted in an higher serum BC concentration in young subjects within 8 and at 24 hours post BC administration, whereas a higher caloric content resulted in a decrease in serum BC concentration in older subjects within 8 hours of BC administration. CONCLUSION: Results indicate that the TGWM provides an accurate means for assessing the intestinal retention of BC in humans.

Expression of mRNA for the gap-junctional protein connexin43 in human colonic tissue is variable in response to beta-carotene supplementation.

Increased expression of the gap-junctional protein connexin43 (Cx43) is reported to be increased in mouse and human dermal fibroblasts in vitro in response to beta-carotene treatment. In the present study, we determined the level of Cx43 mRNA expression in colonic mucosa from normal subjects and subjects with a prior history of colonic polyps or cancer before and after three months of administration of a placebo or beta-carotene. RNA was reverse transcribed and used in a polymerase chain reaction assay employing primers selected from the human Cx43 gene sequence. Cx43 mRNA expression was normalized on the basis of beta 2-microglobulin mRNA expression. The beta-carotene concentration in colonic mucosa, as well as serum and diet, was also determined to establish a correlation between Cx43 expression and beta-carotene concentration. In an initial analysis of samples collected from 10 subjects before supplementation, the quantity of Cx43 mRNA was variable and did not correlate with beta-carotene intake or the concentration of beta-carotene in tissue or serum. In samples collected at zero and three months from eight subjects who were controls or received a placebo, there was no correlation between Cx43 mRNA level and tissue or serum beta-carotene concentration. In samples collected from subjects before and after three months of beta-carotene supplementation, there was a significant increase in tissue and serum beta-carotene concentration in all subjects and an increase in Cx43 mRNA expression after supplementation relative to baseline in four of six samples. The high variability in Cx43 expression indicates that induction of Cx43 mRNA expression is not solely dependent on the concentration of beta-carotene in diet, serum, or tissue. However, the results from subjects supplemented with beta-carotene suggest that induced expression may occur in colonic tissue of some individuals. some individuals.

Variable effects of sodium butyrate on the expression and function of the MDR1 (P-glycoprotein) gene in colon carcinoma cell lines.

Expression of the MDR1 (P-glycoprotein) gene confers resistance to several classes of chemotherapeutic drugs (multi-drug resistance). Colon carcinomas frequently express high levels of MDR1 mRNA and P-glycoprotein, presumably reflecting the origin of these tumors from MDR1-expressing normal colonic cells. In 4 colon carcinoma cell lines (SW 620, HCT-15, DLD-1, LS 180), MDR1 expression was reported in an earlier study to be elevated after exposure to a differentiating agent, sodium butyrate (NaB). In one of these cell lines (SW 620), increased MDR1 expression reportedly was not accompanied by a decrease in the accumulation or cytotoxicity of vinblastine, a P-glycoprotein-transported drug, suggesting a possible functional abnormality of NaB-induced P-glycoprotein. We have re-examined the effect of NaB on MDR1/P-glycoprotein expression and function in the same colon carcinoma cell lines. NaB treatment induced differentiation-related changes and increased expression of MDR1 mRNA in all 4 cell lines. A major increase in MDR1 mRNA and P-glycoprotein expression was observed in only one line, SW 620. This increase, however, was accompanied by a commensurate increase in the activity of P-glycoprotein, indicating that the induced protein was fully functional. NaB treatment caused a relatively minor increase in MDR1 mRNA expressed in the other 3 cell lines. Two of these lines showed a detectable increase in the P-glycoprotein expression and function, but in the third line (LS 180) P-glycoprotein was undetectable either before or after exposure to NaB. The magnitude of MDR1 induction by NaB showed no apparent correlation with differentiation-related changes induced by this agent.

Detection of Porphyromonas gingivalis in oral plaque samples by use of the polymerase chain reaction.

Periodontitis is believed to be caused by bacteria which inhabit periodontal pockets. The identification of these periodontal pathogens by currently available methods requires considerable time and expertise. In this study, we have used a polymerase chain reaction (PCR) assay that is quick, relatively simple, and can detect low numbers of a putative periodontal pathogen. Primers specific for the fimbrial gene of Porphyromonas gingivalis were selected from the published sequence and used for amplification of a 131-basepair sequence of genomic DNA. The PCR detected as few as 100 P. gingivalis cells obtained from pure cultures. Three other bacteria (Actinobacillus actinomycetemcomitans, Prevotella intermedia, and Capnocytophaga gingivalis) that are also putative periodontal pathogens yielded no PCR product at any of the cell concentrations used. This assay was also used for detection of P. gingivalis in subgingival plaque. Five of 13 subgingival bacterial plaque samples obtained from four advanced adult periodontitis patients and two samples from a prepubescent child with advanced periodontitis contained P. gingivalis. The protocol developed is relatively simple and can be completed within four hours of the time of sample acquisition.