Cyclooxygenase-2 expression is abundant in alveolar type II cells in lung cancer-sensitive mouse strains and in premalignant lesions.

Overexpression of cyclooxygenase-2 (COX-2) is seen in a high percentage of human colon tumors, lung adenocarcinomas and other cancers. Inhibition of this enzyme represses human colon tumorigenesis and decreases lung tumor multiplicity in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-exposed A/J mice. The purpose of this investigation was to characterize the expression of cyclooxygenase-2 (COX-2) during tumor progression in the A/J mouse lung and to compare the results with expression in other cancer-susceptible and several cancer-resistant mouse strains. Analysis of normal A/J mouse lung showed that type II alveolar epithelial cells express high levels of COX-2 protein and mRNA, indicating that COX-2 is present constitutively in this tumor progenitor cell prior to any carcinogen exposure. Examination of lung-cancer-resistant (C3H/HeJ, C57BL/6J, DBA/2J) and other lung-cancer-susceptible (A/WySnJ, SWR/J) strains showed similar levels of COX-2 mRNA expression in the three susceptible strains and lower levels of expression in two of the resistant strains, indicating a possible correlation between COX-2 expression in type II cells and lung cancer susceptibility. COX-2 protein expression was observed in A/J lung tumors at all stages of development. Variation and occasional absence of protein expression were also observed in A/J lung tumors, particularly in adenomas and adenocarcinomas, suggesting that COX-2 is not obligatory for maintenance of the malignant phenotype. In support of this conclusion, treatment of xenografted cell lines derived from malignant murine pulmonary tumors with COX-2 inhibitors produced only a slight repression of growth. However, the frequent expression of COX-2 in early lesions in the A/J mouse lung combined with the known reduction in tumor number in animals treated with COX-2 inhibitors prior to carcinogen exposure indicate that COX-2 could be a promising target for lung cancer chemoprevention. In addition, high levels of COX-2 expression in the normal tumor-progenitor cells of lung-cancer-sensitive mice indicate that COX-2 may play a role in lung cancer susceptibility.

Vitamin A-sensitive tissues in transgenic mice expressing high levels of human cellular retinol-binding protein type I are not altered phenotypically.

The suggested function of cellular retinol-binding protein type I [CRBP(I)] is to carry retinol to esterifying or oxidizing enzymes. The retinyl esters are used in storage or transport, whereas oxidized forms such as all-trans or 9-cis retinoic acid are metabolites used in the mechanism of action of vitamin A. Thus, high expression of human CRBP(I) [hCRBP(I)] in transgenic mice might be expected to increase the production of retinoic acid in tissues, thereby inducing a phenotype resembling vitamin A toxicity. Alternatively, a vitamin A-deficient phenotype could also be envisioned as a result of an increased accumulation of vitamin A in storage cells induced by a high hCRBP(I) level. Signs of vitamin A toxicity or deficiency were therefore examined in tissues from transgenic mice with ectopic expression of hCRBP(I). Testis and intestine, the tissues with the highest expression of the transgene, showed normal gross morphology. Similarly, no abnormalities were observed in other tissues known to be sensitive to vitamin A status such as cornea and retina, and the epithelia in the cervix, trachea and skin. Furthermore, hematologic variables known to be influenced by vitamin A status such as the hemoglobin concentration, hematocrits and the number of red blood cells were within normal ranges in the transgenic mice. In conclusion, these transgenic mice have normal function of vitamin A despite high expression of hCRBP(I) in several tissues.

Effect of cigarette smoke on CYP1A1, CYP1A2 and CYP2B1/2 of nasal mucosae in F344 rats.

Enzymes of the nasal tissue, one of the first tissues to contact inhaled toxicants, are relatively resistant to induction by traditional inducers. Because tobacco smoke has been shown to induce cytochrome P450 1A1 (CYP1A1) in rat and human lung tissue, we hypothesized that it would also alter levels of xenobiotic-metabolizing enzymes in nasal mucosae. In the present study, the effect of mainstream cigarette smoke (MCS) on nasal CYP1A1, CYP1A2 and CYP2B1/2 was explored. Four groups of 30 F344 rats were exposed to MCS (100 mg total particulate matter/m3) or filtered air for 2 or 8 weeks. Western analysis of microsomes from nasal tissue of MCS-exposed rats showed an induction of CYP1A1 in respiratory and olfactory mucosae, as well as liver, kidney and lung. Relative to controls, CYP1A2 levels increased slightly in the liver and olfactory mucosa. CYP2B1/2, which increased in the liver, appeared to decrease in upper and lower respiratory tissues. Little to no immunoreactivity with CYP1A1 antibody was observed in fixed nasal sections of control rats, yet intense immunoreactivity was seen in epithelia throughout the nasal cavity of MCS-exposed rats. Ethoxyresorufin O-deethylase activity (associated with CYP1A1/2) decreased approximately 2-fold in olfactory mucosa, but increased in non-nasal tissues of rats exposed to MCS. Methoxy- and pentoxyresorufin O-dealkylase activities (associated with CYP1A2 and CYP2B1/2, respectively) decreased in olfactory and respiratory mucosae, as well as lung (CYP2B1/2), yet increased in liver. These data suggest that xenobiotic-metabolizing enzymines of the nasal mucosae may be regulated differently than other tissues.

Variable expression of cellular retinol- and cellular retinoic acid-binding proteins in the rat uterus and ovary during the estrous cycle.

Ovaries and uteri from normal adult female rats at known stages of the estrous cycle were analyzed for the presence of cellular retinol-binding protein (CRBP) and both types of cellular retinoic acid-binding protein (CRABP and CRABP II). Northern and Western blot analysis of the uteri revealed a peak of CRBP during diestrus and a peak of CRABP during proestrus, whereas CRABP II peaked sharply during estrus. Immunohistochemical studies showed CRABP II localized to the luminal epithelium, while both CRBP and CRABP were observed only in the smooth muscle layers of the uterus. In the ovary, CRABP was not detected, while CRBP levels remained relatively constant throughout the cycle and CRABP II peaked slightly during metestrus. CRBP in the ovary was localized to the oocytes, nearby granulosa cells, and some regions of stroma. CRABP II was found predominantly in the granulosa cells of mature follicles and early corpora lutea, as well as some regions of the stroma. These results suggest a need for further studies to assess the role of retinol and its metabolites in normal uterine function and ovarian follicular development.

Localization of cellular retinoic acid-binding protein (CRABP) II and CRABP in developing rat testis.

Retinoic acid (RA) has been implicated as a signaling molecule for the morphogenesis of some tissues and organs. The morphogenesis of the rat testis occurs relatively late in development, culminating in puberty. Two members of the superfamily of small intracellular carrier proteins for lipophilic compounds are cellular Ra-binding protein (CRABP) and cellular RA-binding protein II (CRABP-II). Both CRABP and CRABP-II are present at various sites in the developing mouse embryo. Here we report the developmental expression and localization of CRABP and CRABP-II in rat testis. Northern blot analysis of CRABP-II demonstrated the highest messenger RNA expression on day 4 (the earliest time point assayed by this technique), decreasing thereafter until day 20, when it became undetectable. Western blot analysis, begun on day 19 of fetal development, indicated that high levels of protein expression in the testis already existed at that time. CRABP messenger RNA expression reached its highest levels between postnatal days 16-20 and decreased thereafter. Immunolocalization revealed that CRABP-II was confined to the fetal population of Leydig and Sertoli cells. We observed that CRABP-II was expressed in certain cells that synthesized retinoic acid in the uterus and ovary (unpublished). The expression of CRABP-II in Sertoli cells and fetal Leydig cells suggested that these cells may well be the site of RA synthesis in the developing testis. CRABP was localized to gonocytes in earlier stages and spermatogonia later, where it was clearly excluded from the nucleus, indicating that the role of CRABP may be to protect these cells from the effects of RA. The reported expression of CRABP-II in embryonal tissues, which are RA responsive and undergoing morphogenesis, coupled with CRABP-II expression in the testis at a critical morphogenic stage suggest that RA may play a prominent role in the morphogenesis of the testis.

Regulation and localization of cellular retinol-binding protein, retinol-binding protein, cellular retinoic acid-binding protein (CRABP), and CRABP II in the uterus of the pseudopregnant rat.

Three members of the superfamily of small intracellular carrier proteins for lipophilic compounds are cellular retinol-binding protein (CRBP), cellular retinoic acid-binding protein (CRABP), and cellular retinoic acid-binding protein II (CRABP II). Retinol-binding protein (RBP) is a secreted protein that binds and solubilizes vitamin A for transport. Here we report the coordinate regulation of RBP, CRBP, retinol, and CRABP II in the uterus of the pseudopregnant rat. In the proliferative stage of the uterus, which was induced by PMSG, the messenger RNA (mRNA) and protein levels of RBP and CRBP as well as retinol levels significantly decreased. This pattern of regulation was duplicated by estrogen treatment of prepubertal rats. In addition, CRBP and RBP were found to be colocalized to the stromal cells of the rat uterus by immunohistochemistry and [35S]methionine-labeled affinity chromatography, respectively, and were not detected in other cell populations. CRABP II mRNA and protein expression were up-regulated in the proliferative phase of the uterus brought about by PMSG injection or, alternatively, by estrogen treatment of prepubertal rats. CRABP II was localized to the surface epithelium, but was not seen elsewhere, including glandular epithelium. Immunolocalization of CRABP showed staining of the smooth muscle and stromal cells of the uterus. The appearance of CRABP in the stroma of the uterus also correlated with PMSG injection as well as estrogen treatment. Although estrogen induced the appearance of both binding proteins, CRABP mRNA levels peaked between 4-24 h postestrogen treatment, whereas CRABP II mRNA levels continued to rise 48 h postestrogen treatment. These data demonstrate an important role for vitamin A and retinoid-binding proteins in rat uterine physiology.

Intestinal vitamin A metabolism: coordinate distribution of enzymes and CRBP(II).

In the mucosal layer of the small intestine, we found nearly identical gradients of CRBP(II), retinal reductase, and LRAT levels down the duodenal-ileal axis, suggesting coordinate regulation of these three proteins. In all cases the level of binding protein or enzyme activity was greatest in the proximal intestine and then decreased sharply in the distal half. This pattern fits with the known capacity of the intestine to absorb vitamin A. In addition, the retinal reductase activity was found predominantly in the intestinal mucosa, while LRAT activity was found in both the intestinal mucosa and muscle. An even distribution of LRAT activity along the longitudinal axis of the intestinal muscle was consistent with an even distribution of CRBP in that tissue. In conjunction with LRAT activity and CRBP, we found endogenous retinyl ester stores in the intestinal muscle layer. The patterns of retinyl ester produced by LRAT in vitro and found in vivo were similar, with retinyl palmitate predominating and a high percentage comprised of retinyl stearate. We also observed a bile salt-independent retinyl ester hydrolase activity in intestinal muscle whose distribution paralleled the retinyl ester stores and LRAT levels. This hydrolase appears to be distinct from retinyl ester hydrolases described from other organs as its activity was insensitive to retinyl ester chain length, the presence of bile salts, or the addition of apo-CRBP. This activity was inhibited by diethyl-p-nitrophenyl-phosphate (IC50 100 microM) and diethylpyrocarbonate (IC50 10 microM), demonstrating a requirement for active serine and histidine residues. In addition, we describe an activity present in some intestinal microsomal preparations that can perturb determinations of reductase and LRAT activity and must be avoided.

Effects of pharmacological retinoids on several vitamin A-metabolizing enzymes.

Fenretinide (HPR), 13-cis-retinoic acid, and all-trans-retinoic acid are vitamin A derivatives used in the treatment of cancer and severe acne. Patients taking these drugs often show side effects resembling the symptoms of hypovitaminosis A, namely, night blindness and decreased plasma retinol levels. A dietary vitamin A deficiency is not suspected in these patients; therefore, interference with normal vitamin A metabolism seems likely. The effect of these drugs on two enzymes involved in vitamin A metabolism was investigated. At micromolar concentrations, all three derivatives were found to inhibit intestinal lecithin-retinol acyltransferase (LRAT) and to a lesser extent liver LRAT and intestinal retinal reductase. Inhibition of intestinal LRAT by HPR and 13-cis-retinoic acid was enhanced by preincubation prior to assay, whereas inhibition of the other activities was not. The Ki for the inhibition of intestinal LRAT by HPR was determined to be 24.1 +/- 5.6 microM. The ability of these drugs to inhibit retinal reduction and retinol esterification in vitro suggests an ability to interfere with normal vitamin A metabolism in vivo, particularly during absorption. This may be most significant for HPR, which is known to accumulate in the liver and intestine after chronic dosing.

NMR relaxation properties of 77Se-labeled proteins.

A 77Se-containing moiety has been attached to cysteine residues in bovine hemoglobin, reduced ribonuclease A, and glutathione by reaction with [77Se]6,6'-diselenobis(3-nitrobenzoic acid). The resultant species contain Se-S linkages that have 77Se NMR absorptions in the range range of 568-580 ppm. Spectra have been recorded at 4.7 and 9.7 tesla (T). For labeled hemoglobin a line width of 250 Hz is seen at 4.7 T and 1000 Hz at 9.4 T. This quadrupling of line width with doubling of observational field strength is consistent with exclusive relaxation by the chemical shift anisotropy (CSA) mechanism. These line widths are greater than expected for a molecule the size of hemoglobin and indicate some aggregation at the high concentrations used. Upon dissociation and partial unfolding of the hemoglobin subunits, the line widths of the selenium resonance decrease to 35 and 120 Hz at 4.7 and 9.4 T, respectively. The spin-lattice relaxation time (T1) for the dissociated hemoglobin at 9.4 T was found to be 220 ms. Together with a value of 377 ms for the spin-spin relaxation time (T2), determined from the line width, an estimate of the CSA was made. This gave a value of 890 ppm, which is in accord with other values for Se(II) linked only by single bonds. When this value for the CSA is used, together with the CSA contribution to the line width, in estimating a correlation time for seleno(3-nitrobenzoic acid) (SeNB)-labeled glutathione, a value of 4 x 10(-11) s is obtained. For SeNB-labeled denatured ribonuclease, four distinct resonances are resolvable at 4.7 T and five resonances at 9.4 T. From T1 values for these resonances and the value of 890 ppm for the CSA, an appropriate correlation time of 0.1 ns was determined, which should result in 77Se resonances of 0.2-1.0 Hz at 4.7 and 9.4 T, respectively. Much greater apparent line widths are observed, which are attributed to microheterogeneity resulting from formation of inter- and intramolecular disulfide linkages. It is concluded that when there are no complications from protein aggregation or chemical exchange, the CSA values anticipated to exist in glutathione peroxidase or other selenoproteins should result in resonances with line widths in the range from 27 to 170 Hz, depending on field strength. These resonances should therefore be observable in the intact protein, if 77Se-enriched material is available.