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To understand the mechanism of retinoid resistance, we studied the subcellular localization and function of retinoid receptors in human breast cancer cell lines. Retinoid X receptor alpha (RXR alpha) localized throughout the nucleoplasm in retinoid-sensitive normal human mammary epithelial cells and in retinoid-responsive breast cancer cell line (MCF-7), whereas it was found in the splicing factor compartment (SFC) of the retinoid-resistant MDA-MB-231 breast cancer cell line and in human breast carcinoma tissue. In MDA-MB-231 cells, RXR alpha was not associated with active transcription site in the presence of ligand. Similarly, ligand-dependent RXR homo- or heterodimer-mediated transactivation on RXR response element or RARE showed minimal response to ligand in MDA-MB-231 cells. Infecting MDA-MB-231 cells with adenoviral RXR alpha induced nucleoplasmic overexpression of RXR alpha and resulted in apoptosis upon treatment with an RXR ligand. This suggests that nucleoplasmic RXR alpha restores retinoid sensitivity. Epitope-tagged RXR alpha and a C-terminus deletion mutant failed to localize to the SFC. Moreover, RXR alpha localization to the SFC was inhibited with RXR alpha C-terminus peptide. This peptide also induced ligand-dependent transactivation on RXRE. Therefore, the RXR alpha C terminus may play a role in the intranuclear localization of RXR alpha. Our results provide evidence that altered localization of RXR alpha to the SFC may be an important factor for the loss of retinoid responsiveness in MDA-MB-231 breast cancer cells.

Lung cancer is the world's leading cause of cancer death. Since progress in the treatment of this cancer has been exceedingly slow, the upswing in tobacco consumption in many sectors becomes even more tragic. One area for cautious optimism is the recent pilot reports of improved early lung cancer detection using new spiral CT techniques from institutions in Japan and New York. The prospect of improved early detection in a major cancer raises a number of public health concerns and highlights the importance of critical validation of this proposed new tool. From experience with early detection-based management of other cancers, it is evident that the entire process of detection, case validation, intervention, monitoring and public education needs to be carefully developed. The International Association for the Study of Lung Cancer has worked with the National Cancer Institute over the last decade to nurture interest and expertise in conducting population-based management of early lung cancer. A distillation of this process up to the current time is reviewed in this manuscript.

Neurofibromin is a tumor suppressor protein, which is similar in function to the GTPase activating protein (GAP), p120GAP, in that it accelerates inactivation of Ras. Mutations in the NF1 gene cause neurofibromatosis type 1, NF1, an autosomal dominant disease with a diverse spectrum of clinical manifestations, including neurofibromas. Ras activation (GTP binding) is induced by the GTP exchange factor Sos and its inactivation is regulated through the GAPs (p120GAP and neurofibromin). Strikingly, neurofibromin was nearly absent in MB-231 human breast cancer cells and present in the remaining four cell lines studied, with higher levels in BT-474 and MB-453 than in MCF-7 and BT-20 cells, as tested with polyclonal antibodies to both the N-terminal as well as the C-terminal peptides. Coordinated with the near absence of neurofibromin, these cells also presented with much greater levels of P-MAPK and activated Ras. Further, RT-PCR analysis demonstrated the absence of expression of NF1 mRNA type I isoform only in the MB-231 cell lines. This result documents for the first time an altered NF1 expression at the protein and mRNA levels in MDA-MB-231 breast cancer cells.

Most studies have reported an up-regulation of retinoic acid receptor (RAR) mRNA expression by all-trans retinoic acid (RA). We aimed to study the effect of RA on RAR protein levels in MCF-7 human breast cancer cells. Incubation of these cells with 10(-6) M RA induced a rapid breakdown of both RARalpha and RARgamma in spite of the accumulation of their mRNAs. Proteasome specific inhibitors blocked the RA-induced breakdown of RARs. Furthermore, RA enhanced the formation of the complex between RARalpha and ubiquitin in a concentration- and time-dependent manner, suggesting the involvement of ubiquitin and proteasome in this reaction. Retinoid X receptor alpha (RXRalpha) was also decreased, albeit to a lesser extent, in RA-treated cells. Use of synthetic receptor agonists and antagonists clearly showed that the effect of the retinoid on the breakdown of the retinoid receptors is receptor-ligand agonist-dependent and blunted by the antagonist. An electrophoretic mobility shift assay, using nuclear extracts from RA-treated cells, showed that a reduction in complex formation with hormone response elements correlated with the reduction of RAR and RXR protein. These data suggest that RA induces the breakdown of RARs through a process involving ubiquitination and that this phenomenon causes a reduction in the formation of DNA-receptor complexes.

Retinyl ester concentration is regulated by retinoic acid (RA) through an autoregulatory loop, which acts on lecithin:retinol acyltransferase (LRAT). We tested whether retinol esterification activity is downregulated in human mammary carcinoma cells and whether LRAT expression is RAR-regulated. Normal human mammary epithelial (HMEC) cells expressed a retinoid-upregulated 5-kb LRAT transcript and synthesized retinyl esters from 3H-retinol. Human carcinoma MCF-7 cells failed to express the 5-kb LRAT transcript and to synthesize retinyl esters. Instead, they expressed a 2.7-kb LRAT transcript. Both transcripts were upregulated by RA. Stable expression of the dominant-negative RARalpha403 blunted the up-regulation of LRAT mRNA by RA. We conclude that retinol esterification is decreased in MCF-7 vs normal mammary cells; that these cancer cells express a shorter (2.7 kb) LRAT transcript, and that retinoid receptors are involved in the regulation of LRAT-mediated retinyl ester synthesis by RA.

The current mortality rate for lung cancer exceeds 85%, as it has for the last 3 decades. This statistic reflects the utility of the major diagnostic tool that has been used during this period to diagnose lung cancer: the chest X-ray. The overwhelming majority of new cases of lung cancer that are detected with chest X-rays involve individuals who already have regional or distant metastatic disease. Because the systemic treatment of this disease has not improved greatly, patients with metastatic disease rarely are cured. This article reviews the issues involved with the development of sputum-based cellular diagnostics for early stage lung cancer. The biomarker, heterogeneous nuclear ribonucleoprotein A2/B1, is the lead marker for this approach. It has been used in several studies in independent cohorts that have suggested that its overexpression in bronchial epithelial cells is associated highly with the development of lung cancer. This marker is detectable 1 year or more prior to the detection of lung cancer by chest X-ray. Finding this early airway-confined phase of lung cancer may allow for the evolution of new management approaches for very early stage lung cancer. Research activities, such aerosolized chemoprevention, are discussed.

Chemopreventive retinoids may be more effective if delivered to the lung epithelium by inhalation. 13-cis-Retinoic acid (13-cis-RA) was comparable to all-trans-retinoic acid (RA) in inducing transglutaminase II (TGase II) in cultured human cells. Inhaled 13-cis-RA had a significant stimulatory activity on TGase II in rat lung (P < 0.001) but not in liver tissue (P < 0.544). Furthermore, inhaled 13-cis-RA at daily deposited doses of 1.9 mg/kg/day up-regulated the expression of lung retinoic acid receptors (RARs) alpha, beta, and gamma at day 1 (RARalpha by 3.4-fold, RARbeta by 7.2-fold, and RARgamma by 9.7-fold) and at day 17 (RARalpha by 4.2-fold, RARbeta by 10.0-fold, and RARgamma by 12.9-fold). At a lower aerosol concentration, daily deposited doses of 0.6 mg/kg/day were also effective at 28 days. Lung RARalpha was induced by 4.7-fold, RARbeta by 8.0-fold, and RARgamma by 8.1-fold. Adjustment of dose by exposure duration was also effective; thus, inhalation of an aerosol concentration of 62.2 microg/liter, for durations from 5 to 240 min daily for 14 days, induced all RARs from 30.6- to 74-fold at the shortest exposure time. None of the animals exposed to 13-cis-RA aerosols showed RAR induction in livers. By contrast, a diet containing pharmacological RA (30 microg/g of diet) failed to induce RARs in SENCAR mouse lung, although it induced liver RARs (RARalpha, 21.8-fold; RARbeta, 13.5-fold; RARgamma, 12.5-fold); it also failed to induce lung TGase II. A striking increase of RARalpha expression was evident in the nuclei of hepatocytes. Pharmacological dietary RA stimulated RARalpha, RARbeta, and RARgamma as early as day 1 by 2-, 4-, and 2.1-fold, respectively, without effect on lung RARs. Therefore, 13-cis-RA delivered to lung tissue of rats is a potent stimulant of lung but not liver RARs. Conversely, dietary RA stimulates liver but not lung RARs. These data support the concept that epithelial delivery of chemopreventive retinoids to lung tissue is a more efficacious way to attain up-regulation of TGase II and the retinoid receptors and possibly to achieve chemoprevention.

In previously treated head-and-neck cancer patients, p.o. administered isotretinoin (13-cis retinoic acid) reduced the occurrence of second aerodigestive tumors, including lung tumors, but side effects made chronic therapy problematic. We reasoned that inhaled isotretinoin might provide sufficient drug to the target cells for efficacy while avoiding systemic toxicity, and we proceeded with the pilot study reported here. Male A/J mice were given single i.p. doses of urethane, a common experimental lung carcinogen, or benzo[a]pyrene (BaP) or 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), putative major carcinogens in tobacco smoke. The following day, exposures to isotretinoin aerosols for 45 min daily at 1.3, 20.7, or 481 microg/l were initiated. After 2 weeks, the high dose caused severe toxicity on the snout skin, necessitating a reduction of dose frequency to twice a week. As a precaution, the mid dose was reduced to three exposures per week. The weekly total deposited doses after the dose frequency reductions were calculated to be 0.24, 1.6, and 24.9 mg/kg for the low, mid, and high doses, of which 16% was estimated to be deposited in the lungs. The weekly deposited pulmonary drug doses were calculated to be 0.01, 0.07, and 1.1% of a previously reported ineffective oral dose in urethane-treated A/J mice. After 10-16 weeks, mice were sacrificed to count areas of pulmonary hyperplasia and adenomas. For all carcinogens, the mice exposed to the high isotretinoin dose showed reductions of tumor multiplicity ranging from 56 to 80% (P < 0.005). The mid dose was associated with reductions of tumor multiplicity by 67 and 88% (P < 0.005) in BaP- and NNK-treated mice, respectively, and was tolerated until approximately 12 weeks, when both these and the high-dose mice began losing weight. The low-dose mice had nonsignificant reductions of 30% (P < 0.13) and 16% (P < 0.30) for BaP- and NNK-treated mice, respectively without any evidence of side effects. For BaP- and NNK-treated mice, numbers of hyperplastic areas directly correlated to dose level and inversely to tumor number, suggesting arrested progression. Inhaled mid-dose isotretinoin caused up-regulation of lung tissue nuclear retinoic acid receptors (RARs) relative to vehicle-exposed mice, RARalpha (3.9-fold vehicle), RARbeta (3.3-fold), and RARgamma (3.7-fold), suggesting that these receptors may be useful biomarkers of retinoid activity in this system. The encouraging results from this pilot study suggest that inhaled isotretinoin merits evaluation in people at high risk for lung cancer.

The key role of vitamin A in embryonal development is reviewed. Special emphasis is given to the physiological action of retinoids, as evident from the retinoid ligand knockout models. Retinoid metabolism in embryonic tissues and teratogenic consequences of retinoid administration at high doses are presented. Physiological and pharmacological actions of retinoids are outlined and explained on the basis of their interactions as ligands of the nuclear retinoid receptors. Immediate target genes and the retinoid response elements of their promoters are summarized. The fundamental role of homeobox genes in embryonal development and the actions of retinoids on their expression are discussed. The similarity of the effects of retinoid ligand knockouts to effects of compound retinoid receptor knockouts on embryogenesis is presented. Although much remains to be clarified, the emerging landscape offers exciting views for future research.

Annexin V is a Ca2+-dependent phospholipid binding protein. Although it has been shown to inhibit protein kinase C (PKC) in cell-free systems, its role in the intact cell is unclear. A stable MCF-7 human breast cancer cell overexpression system was established to investigate the function of annexin V. In these cells, 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced phosphorylation and kinase activity of ERK1/2 were suppressed. Morphological changes induced by TPA were reduced by annexin V overexpression as well as by the pan-PKC inhibitor, bisindolylmaleimide I, and by the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) inhibitor, PD98059. TPA-induced MEK1/2 and Raf-1 phosphorylation were reduced in these cells. The TPA-enhanced active Ras, and its association with Raf-1, were reduced. TPA treatment of MCF-7 cells caused an increased association of Shc with Grb2. However, this increased association was prevented in the annexin V-overexpressors. p21WAF/CIP1 is responsible for inhibition of cell cycle progression in MCF-7 cells. TPA induced the expression of p21WAF/CIP1 to a greater extent in MCF-7 parent and control plasmid cells than in annexin V overexpressors. PD98059 inhibited this increase, suggesting that TPA upregulation of p21WAF/CIP1 occurs via the MEK pathway, and that annexin V overexpression blunts it. This work shows that annexin V overexpression suppresses the TPA-induced Ras/ERK signaling by inhibiting at/or upstream of Shc, possibly through the inhibition of PKCs. Oncogene (2000).

To examine the role of retinoids in hematopoietic cell growth in vivo, we studied female SENCAR mice made vitamin A deficient by dietary restriction. Deficient mice exhibited a dramatic increase in myeloid cells in bone marrow, spleen, and peripheral blood. The abnormal expansion of myeloid cells was detected from an early stage of vitamin A deficiency and contrasted with essentially normal profiles of T and B lymphocytes. This abnormality was reversed on addition of retinoic acid to the vitamin A-deficient diet, indicating that the myeloid cell expansion is a direct result of retinoic acid deficiency. TUNEL analysis indicated that spontaneous apoptosis, a normal process in the life cycle of myeloid cells, was impaired in vitamin A-deficient mice, which may play a role in the increased myeloid cell population. Quantitative reverse transcriptase-polymerase chain reaction analysis of purified granulocytes showed that expression of not only RAR, but RXRs, 2 nuclear receptors that mediate biologic activities of retinoids, was significantly reduced in cells of deficient mice. This work shows that retinoids critically control the homeostasis of myeloid cell population in vivo and suggests that deficiency in this signaling pathway may contribute to various myeloproliferative disorders.

Purpose: Our previous work had shown that retinoic acid (RA) inhibits cell growth and induces apoptosis in estrogen receptor-positive (ER-positive) MCF-7 and T-47D human breast carcinoma cells, but not in ER-negative human breast carcinoma cells MB-231 and MB-453. The purpose of this work was to determine whether these differences might be due to differences in uptake and metabolism of the drug between ER-positive and ER-negative cells., Methods: We measured RA uptake in cultured human breast cancer cells and determined its metabolism by high-pressure liquid chromatographic analysis., Results: The two ER-positive cell lines reached maximum RA uptake at about 2 h, followed by a sharp decline, so that most RA had disappeared from the cells and from the medium by 24 h and was found as oxidation products in the culture medium. In contrast, the two ER-negative cell lines showed a pattern of lower accumulation without the sharp increase and subsequent steep decline, so that by 24 h there was more RA in these cells and their culture medium than in the RA-responsive ER-positive cells, even though at 2 h the ER-negative cells had taken up less RA than the ER-positive cells. Kinetic analysis of the uptake of RA in MCF-7 cells was consistent with rapid movement across the cell membranes and the actual rate determined by diffusion of albumin-bound retinoid to the cells., Conclusions: This study is the first to demonstrate profound differences in RA accumulation and confirms previous results on different rates of RA metabolism between ER-positive and ER-negative human breast cancer cells. The findings reported here, therefore, may introduce additional elements to be considered in the design of new drugs for cancer chemoprevention and therapy.

Clinical trials have shown a significant increase in incidence of lung cancer among smokers and asbestos workers supplemented with beta-carotene, suggesting a tumor-promoting activity for this agent. We set out to test possible tumor-promoting and chemopreventive activities of dietary and topical beta-carotene in the two-stage 7,12-dimethylbenz[a]anthracene-12-O-tetradecanoylphorbol 13-acetate (TPA) model of mouse skin carcinogenesis. In the first study, the effects of three levels of dietary beta-carotene (6, 60, and 600 micrograms/g purified diet containing no other retinoid or carotenoid) were assessed over a period of 42 weeks. Carcinoma yield was reduced by approximately 50% in the 600 micrograms/g diet group (mean 0.22 carcinomas/mouse) compared with the 6 micrograms/g diet group (mean 0.44 carcinomas/mouse, p = 0.003). The 60 micrograms/g diet group showed a pattern of inhibition similar to the 600 micrograms/g diet group. A protective effect (25% reduction) of beta-carotene (in the 600 micrograms/g diet group) on papilloma formation was also found. However, the intermediate 60 micrograms/g diet group showed the same incidence as the low 6 micrograms/g diet group. This points to a lack of correlation between papilloma and carcinoma incidence, as we also found in previous work on dietary retinoids and carotenoids. The purpose of the second study was to assess whether topical beta-carotene (2 micrograms) has tumor-promoting or chemopreventive activity in the two-stage protocol. In the absence of TPA, beta-carotene had no significant tumor-promoting activity. Instead, papilloma yield induced by TPA was decreased by topical beta-carotene from an average of 20 to approximately 10 papillomas/mouse (p = 2.5 x 10(-7)). The effect of topical beta-carotene persisted beyond the treatment period (Week 24) until the termination of the study at Week 42. Western blot analysis of mouse skin extracts showed that topical beta-carotene upregulated retinoic acid receptor-alpha and -gamma expression in the dorsal skin. This finding suggests that beta-carotene may work as a chemopreventive agent by upregulating the expression of retinoid receptors in mouse skin. In conclusion, our data show that beta-carotene prevents skin carcinoma formation, induces retinoic acid receptor expression, and fails to act as a tumor promoter in the two-stage model of skin tumorigenesis.

Background: Retinoic acid is necessary for the growth and differentiation of organisms and exerts its molecular actions by binding to specific nuclear receptors that belong to the thyroid-steroid hormone receptor superfamily. Steroids and retinoids control the differentiation of the female reproductive epithelia: estrogen maintains the squamous differentiation of vaginal and ectocervical epithelia, whereas retinoic acid maintains the simple columnar endocervical and uterine epithelia. These lining epithelia transform into a squamous metaplastic phenotype in vitamin A-deficient animals. Furthermore, mortality due to vitamin A deficiency is usually attributed to infection resulting in part from dysfunction of the protective epithelia., Objective: Our objective was to test the hypothesis that estrogen depletion might change the squamous metaplastic response to vitamin A deficiency and affect animal survival., Design: We used female SENCAR mice maintained on a purified vitamin A-deficient diet containing either 0 or 3 microg retinoic acid/g diet. Mice were either ovariectomized or intact. Squamous cells arising in the normally simple columnar epithelium of the endocervix and uterine cavity were monitored by keratin 5 expression with immunohistochemistry., Results: Ovariectomy did not change the time to onset of vitamin A deficiency. It increased the number of squamous metaplastic cells and prolonged survival in mice consuming a vitamin A-deficient diet by as much as 40%., Conclusions: Factors other than epithelial differentiation per se control survival outcome of vitamin A-deficient mice. The results also show a significant increase in longevity of vitamin A- deficient mice when ovariectomized.

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.

Retinoic acid (RA)-resistant cell lines are highly malignant. To inhibit the growth of the RA-resistant cells we used 4-HPR, a synthetic retinoid, which may act through alternative signal transduction pathways. 4-HPR induced cell growth inhibition and apoptosis in all RA-sensitive as well as -resistant cells, demonstrating a wider spectrum of potency over RA. 4-HPR induced tissue TGase activity. A tight correlation between the induction of tissue TGase, the inhibition of cell growth, and apoptosis was evident in all eight RA-sensitive cell lines. However, basal TGase differed in the different cells, suggesting inducibility rather than basal levels as the relevant parameter. In sharp contrast to the RA-sensitive cells, RA-resistant cells showed sporadic response to 4-HPR for tissue TGase. The wider spectrum of activity of 4-HPR in inhibiting cell growth and inducing apoptosis makes it a good candidate for the treatment of RA-resistant cancer cells.

Treatment of estrogen receptor (ER)-positive MCF-7 human breast cancer cells with retinoic acid (RA) inhibited cell growth and increased cell adhesion to fibronectin. In contrast, ER- MDA-MB-231 cells failed to respond. Western blot analysis showed that tyrosine phosphorylation of two major bands at Mr 125,000 and Mr 68,000 was induced by RA in ER+ MCF-7 human breast carcinoma cells. However, this induction was a late phenomenon detectable at 12 and 24 h, but not within 3 h. A similar increase of tyrosine phosphorylation by RA was observed in ER+ human breast cancer cell lines T-47D and ZR-75-1, but not in the ER- cell lines MDA-MB-231, MDA-MB-453, and MDA-MB-468. Focal adhesion kinase and paxillin, which localize in focal adhesion plaques and may play important roles in the integrin signaling pathway, were identified as the major proteins showing RA-induced tyrosine phosphorylation. The retinoid X receptor-selective compound SR11237 failed to induce tyrosine phosphorylation, indicating that retinoid X receptor activation is not involved in this phenomenon. In contrast, stable overexpression of a truncated RA receptor (RAR) alpha cDNA, RARalpha403, with strong RAR dominant negative activity prevented the increase in tyrosine phosphate, suggesting that RAR signaling is involved in RA-induced tyrosine phosphorylation. Tyrosine phosphorylation was induced the most by the RAR-alpha (193836), followed by RAR-gamma (194433), but was not significantly induced by RAR-gamma (193174)-selective retinoids. This study demonstrates a coordinated albeit relatively late effect of RA on cell adhesion and tyrosine phosphorylation in ER+ human breast cancer cells and suggests RAR-alpha as the major responsible retinoid receptor.