The rexinoid, bexarotene, prevents the development of premalignant lesions in MMTV-erbB2 mice.

Retinoids, vitamin A analogues that bind to retinoic acid receptor (RAR) or retinoid X receptor (RXR), play important roles in regulating cell proliferation, apoptosis, and differentiation. Recently, RXR-selective ligands, also referred to as rexinoids, have been investigated as potential chemopreventive agents for breast cancer. Our previous studies demonstrated that the rexinoid bexarotene significantly prevented ER-negative mammary tumourigenesis with less toxicity than naturally occurring retinoids in animal models. To determine whether bexarotene prevents cancer at the early stages during the multistage process of mammary carcinogenesis, we treated MMTV-erbB2 mice with bexarotene for 2 or 4 months. The development of preinvasive mammary lesions such as hyperplasias and carcinoma-in-situ was significantly inhibited. This inhibition was associated with reduced proliferation, but no induction of apoptosis. We also examined the regulation of a number of rexinoid-modulated genes including critical growth and cell cycle regulating genes using breast cell lines and mammary gland samples from mice treated with rexinoids. We showed that two of these genes (DHRS3 and DEC2) were modulated by bexarotene both in vitro and in vivo. Identification of these rexinoid-modulated genes will help us understand the mechanism by which rexinoid prevents cancer. Such rexinoid-regulated genes also represent potential biomarkers to assess the response of rexinoid treatment in clinical trials.

Mapping of epidermal growth factor-, serum-, and phorbol ester-responsive sequence elements in the c-jun promoter.

Expression of the nuclear proto-oncogene c-jun is rapidly and transiently induced by many growth factors, serum, and tumor promoters. The sequence elements in the c-jun promoter involved in serum or growth factor induction have not been identified. The c-jun promoter region between -117 and -72 contains binding sites for the transcription factors Sp1, CTF, and AP-1. An additional sequence element has been noted at position -59. This A+T-rich sequence, formerly proposed as a TFIID-binding site, conforms to the consensus binding sequence of a recently identified factor, RSRF (related to serum response factor). In this study, we mapped the sequences in the c-jun promoter responsible for epidermal growth factor (EGF), serum, and 12-O-tetradecanoylphorbol-13-acetate (TPA) induction by deletion and point mutational analysis. We found that the c-jun RSRF site is an important element for EGF and serum induction of the promoter and that there are several factors in HeLa nuclear extracts which specifically bind to this site. The RSRF site was also sufficient for EGF, serum, and TPA induction when assayed on a heterologous promoter. The c-jun AP-1 site was not required for EGF, serum, or TPA induction but was sufficient to mediate a weak response to these agents when assayed on a heterologous promoter. Double mutation of the RSRF and AP-1 sites suggests that there is an additional TPA-responsive element between -80 and +150 in the c-jun promoter.

mXBP/CRE-BP2 and c-Jun form a complex which binds to the cyclic AMP, but not to the 12-O-tetradecanoylphorbol-13-acetate, response element.

Proto-oncogene products c-Fos and c-Jun form a complex which binds with high affinity to the 12-O-tetradecanoylphorbol-13-acetate (TPA) response DNA element and which stimulates transcription of phorbol ester- inducible genes. We have previously identified, by screening a lambda gt11 expression library, murine protein mXBP, which binds to a sequence which overlaps the 3' end of the murine class II major histocompatibility complex A alpha gene X box, a conserved transcription element found upstream of all class II genes. Here, we demonstrate that the target sequence for mXBP is a consensus cyclic AMP response element (CRE). mXBP is a member of the leucine zipper family of DNA-binding proteins and has significant homology to oncoproteins c-Fos and c-Jun. The inferred amino acid sequence of mXBP shows near identity to human CRE-BP1, except it does not contain an internal proline-rich domain. Immunoprecipitation and glutaraldehyde cross-linking studies show that mXBP/CRE-BP2 can form a complex with c-Jun. Complex formation is dependent on intact leucine zipper domains in both proteins. mXBP-c-Jun complexes can coexist with c-Fos-c-Jun complexes and can bind with high affinity to CRE, but not to TPA response DNA element, sequences. These results suggest that changes in the expression of mXBP/CRE-BP2, c-Fos, and c-Jun, which alter the ratio of mXBP-c-Jun to c-Fos-c-Jun complexes, would affect the relative expression of cyclic AMP and phorbol ester-responsive genes. This provides support for a combinatorial model of gene regulation, whereby protein-protein interactions which alter the DNA binding specificity of protein complexes can expand the flexibility of cellular transcriptional responses.

Effect of the retinoid X receptor-selective ligand LGD1069 on mammary carcinoma after tamoxifen failure.

BACKGROUND: We have previously shown that a retinoid X receptor (RXR)-selective ligand (a rexinoid), called LGD1069, is highly efficacious in both the chemoprevention and the chemotherapy for N-nitrosomethylurea-induced rat mammary carcinomas. To evaluate a possible role for rexinoids in breast cancer therapy further, we have designed and characterized a novel carcinogen-induced model to mimic the clinical situation in which the tumors of patients stop responding to tamoxifen therapy and develop resistance to this drug. METHODS: Rats with experimentally induced mammary tumors were treated with tamoxifen to select a population with primary tumors that failed to respond completely to the drug. Once the failure of tamoxifen therapy had been established, LGD1069 was added to the treatment regimen, and the tumors in these animals were compared with tumors in a group of animals that remained on tamoxifen alone. RESULTS: LGD1069 in combination with tamoxifen for up to 20 weeks yielded an overall objective response rate of 94% (95% confidence interval [CI] = 86%-100%) (includes complete and partial responses) in primary tumors compared with a rate of 33% (95% CI = 11%-56%) in primary tumors treated with tamoxifen alone, a statistically significant difference (two-sided P<.0001). In addition, the LGD1069 and tamoxifen combination was associated with a statistically significant decrease in total tumor burden (two-sided P =.03). In a second study, tumors that failed to respond to tamoxifen therapy exhibited a 51% (95% CI = 34%-71%) objective response rate when treated with LGD1069 alone for 6 weeks after tamoxifen therapy was withdrawn. CONCLUSION: We have demonstrated that the RXR-selective ligand LGD1069 in combination with tamoxifen is a highly efficacious therapeutic agent for tumors that fail to respond completely to tamoxifen. This finding suggests that rexinoid therapy offers a novel approach to the treatment of breast tumors that may have developed resistance to antihormonal therapies such as tamoxifen.

Arsenic trioxide as an inducer of apoptosis and loss of PML/RAR alpha protein in acute promyelocytic leukemia cells.

BACKGROUND: Retinoids, which are derivatives of vitamin A, induce differentiation of acute promyelocytic leukemia (APL) cells in vitro and in patients. However, APL cells develop resistance to retinoic acid treatment. Arsenic trioxide (As2O3) can induce clinical remission in patients with APL, including those who have relapsed after retinoic acid treatment, by inducing apoptosis (programmed cell death) of the leukemia cells. In this study, we investigated the molecular mechanisms by which As2O3 induces apoptosis in retinoic acid-sensitive NB4 APL cells, in retinoic acid-resistant derivatives of these cells, and in fresh leukemia cells from patients. METHODS: Apoptosis was assessed by means of DNA fragmentation analyses, TUNEL assays (i.e., deoxyuridine triphosphate labeling of DNA nicks with terminal deoxynucleotidyl transferase), and flow cytometry. Expression of the PML/RAR alpha fusion protein in leukemia cells was assessed by means of western blotting, ligand binding, and immunohistochemistry. Northern blotting and ribonuclease protection assays were used to evaluate changes in gene expression in response to retinoic acid and As2O3 treatment. RESULTS AND CONCLUSIONS: As2O3 induces apoptosis without differentiation in retinoic acid-sensitive and retinoic acid-resistant APL cells at concentrations that are achievable in patients. As2O3 induces loss of the PML/RAR alpha fusion protein in NB4 cells, in retinoic-acid resistant cells derived from them, in fresh APL cells from patients, and in non-APL cells transfected to express this protein. As2O3 and retinoic acid induce different patterns of gene regulation, and they inhibit the phenotypes induced by each other. Understanding the molecular basis of these differences in the effects of As2O3 and retinoic acid may guide the clinical use of arsenic compounds and provide insights into the management of leukemias that do not respond to retinoic acid.

Induction of adipocyte-specific gene expression is correlated with mammary tumor regression by the retinoid X receptor-ligand LGD1069 (targretin).

Targretin (LGD1069; a high-affinity ligand for the retinoid X receptors) is an efficacious chemotherapeutic and chemopreventive agent in the N-nitroso-N-methylurea-induced rat mammary carcinoma model. To evaluate the molecular action of LGD1069 in mammary carcinoma we have examined gene expression patterns in controls and nonresponding tumors compared with tumors undergoing regression (responding) by LGD1069. When compared with controls or nonresponding tumors, the expression of adipocyte-related genes such as adipocyte P2 (aP2), adipsin, peroxisome proliferator-activated receptor gamma (PPARgamma), and lipoprotein lipase was elevated in LGD1069-responding tumors. Further analysis showed that gene expression changes occurred rapidly, in as little as 6 h, after the first dose of LGD1069. Immunohistochemical analysis showed that aP2 protein was also highly expressed in responding tumors when compared with control or nonresponding tumors. More importantly, aP2 protein was localized in the tumor cells in addition to the adipocytes present in the tumors. Similar changes in gene expression and inhibition in growth were seen in tumor cells (cloned from N-nitroso-N-methylurea-induced carcinoma) exposed to LGD1069 in vitro. These data suggest that tumor regression by LGD1069 involves differentiation induction along the adipocyte lineage.

Beyond tamoxifen: the retinoid X receptor-selective ligand LGD1069 (TARGRETIN) causes complete regression of mammary carcinoma.

Recently, we reported that LGD1069, a high-affinity ligand for the retinoid X receptors (RXRs), was shown to have an efficacy equivalent to that of tamoxifen (TAM) as a chemopreventive agent in the N-nitroso-N-methylurea-induced rat mammary carcinoma model. Furthermore, LGD1069 was very well tolerated during 13 weeks of chronic therapy with no classic signs of "retinoid-associated" toxicities. Due to the high efficacy and benign profile of this RXR agonist as a suppressor of carcinogenesis, we examined its role as a therapeutic agent on established mammary carcinomas. In the rat mammary carcinoma model, N-nitroso-N-methylurea was used to induce tumors, and the tumors were allowed to grow to an established size prior to initiation of treatment. LGD1069-treated animals showed complete regression in 72% of treated tumors and had a reduced tumor load compared to control. In addition, the combination of LGD1069 and TAM showed increased efficacy over either agent alone. Histopathological analysis showed a reduction of LGD1069-treated tumor malignancy, an increase in differentiation, and a sharp decrease in cellular proliferation compared to vehicle-treated control tumors. These data demonstrate that the RXR-selective ligand LGD1069 is a highly efficacious therapeutic agent for mammary carcinoma and enhances the activity of TAM.

Estrogen receptor expression activates the transcriptional and growth-inhibitory response to retinoids without enhanced retinoic acid receptor alpha expression.

Estrogen receptor (ER)-positive human breast cancer cells are hormonally regulated and are inhibited by retinoids, whereas most ER-negative breast cancer cells are not. Here, we compared retinoid-induced transcriptional activation and growth inhibition in the ER-negative breast cancer cell line MDA-MB-231, stably transfected to express wild-type ER (S30), with that of the ER-positive MCF-7 line and the ER-negative parental line. Retinoids inhibited growth of the ER-expressing S30 clone but not of the parental MDA-MB-231 cells. Unlike a previously reported MDA-MD-231 subclone that was transfected to express a mutated ER (G400V), S30 did not express increased levels of retinoid receptor RNA or protein, nor was there increased binding activity to retinoid-responsive DNA elements. However, stable expression of ER increased retinoid activation of transcription of a retinoic acid (RA) response elements from the low level in MDA-MB-231 to approach the level of MCF-7. The restored growth inhibition and transcriptional regulation by RA were unaffected by treatment with ER agonists or antagonists. Transient expression of ER but not of other nuclear receptors in MDA-MB-231 cells also activated retinoid-induced transcription, showing that this response is specific to ER. Furthermore, the effect of exogenously expressed ER on retinoid response was much greater than that obtained by overexpression of RA receptor alpha and/or retinoid X receptor alpha. Finally, a panel of ER mutants showed that enhancement of retinoid-induced transcriptional activity was dependent on the integrity of the DNA binding domain.

Chemoprevention of mammary carcinoma by LGD1069 (Targretin): an RXR-selective ligand.

Recently, 9-cis retinoic acid, a high affinity ligand for retinoic acid receptors and retinoid X-receptors (RXRs), was shown to have efficacy superior to all-trans retinoic acid as a chemopreventive agent in the N-nitroso-N-methylurea-induced rat mammary carcinoma model. To further explore the specific contribution RXR activation may play in suppression of carcinogenesis, the efficacy of LGD1069 (Targretin), an RXR-selective ligand, in the N-nitroso-N-methylurea-induced rat mammary tumor model was studied. LGD1069-treated animals showed a 90% reduction in tumor burden and tumor incidence compared with vehicle-treated rats with an efficacy similar to that achieved with tamoxifen. LGD1069 was very well tolerated during 13 weeks of chronic therapy with no classic signs of "retinoid-associated" toxicities. These data demonstrate that LGD1069, an RXR-selective ligand, can act as a highly effective and benign chemopreventive agent for mammary carcinoma.

A novel retinoic acid receptor-selective retinoid, ALRT1550, has potent antitumor activity against human oral squamous carcinoma xenografts in nude mice.

We have identified a novel retinoid, ALRT1550, that potently and selectively activates retinoic acid receptors (RARs). ALRT1550 binds RARs with Kd values of approximately equal to 1-4 nM, and retinoid X receptors with low affinities (Kd approximately equal to 270-556 nM). We studied the effects of ALRT1550 on cellular proliferation in squamous carcinoma cells. ALRT1550 inhibited in vitro proliferation of UMSCC-22B cells in a concentration-dependent manner with an IC50 value of 0.22 +/- 0.1 (SE) nM. 9-cis-Retinoic acid (ALRT1057), a pan agonist retinoid that activates RARs and retinoid X receptors, inhibited proliferation with an IC50 value of 81 +/- 29 nM. In vivo, as tumor xenografts in nude mice, UMSCC-22B formed well-differentiated squamous carcinomas, and oral administration (daily, 5 days/week) of ALRT1550, begun 3 days after implanting tumor cells, inhibited tumor growth by up to 89% in a dose-dependent manner over the range of 3-75 micrograms/kg. ALRT1550 (30 micrograms/kg) also inhibited growth of established tumors by 72 +/- 3% when tumors were allowed to grow to approximately equal to 100 mm3 before dosing began. In comparison, 9-cis retinoic acid at 30 mg/kg inhibited growth of established tumors by 73 +/- 5%. Interestingly, retinoids did not appear to alter tumor morphologies in UMSCC-22B tumors. Notably, ALRT1550 produced a therapeutic index of approximately equal to 17 in this model, indicating a separation between doses that inhibited tumor growth and that induced symptoms of hypervitaminosis A. In summary, ALRT1550 potently inhibits cellular proliferation in vitro and in vivo in this squamous cell carcinoma tumor model. These data support additional study of ALRT1550 for its potential for improving anticancer therapy in human clinical trials.

Abrogation of transforming growth factor-alpha/epidermal growth factor receptor autocrine signaling by an RXR-selective retinoid (LGD1069, Targretin) in head and neck cancer cell lines.

Clinical studies have demonstrated that retinoids, including retinol (Vitamin A) and its synthetic derivatives, can eradicate leukoplakia and suppress the formation of squamous cell carcinoma of the head and neck (SCCHN). Nonselective retinoids have been shown to abrogate transcriptional activation of transforming growth factor-alpha (TGF-alpha) and epidermal growth factor receptor (EGFR), which characterize SCCHN. LGD1069 (Targretin) is a potent RXR-selective retinoic acid agonist with a reduced toxicity profile compared with other nonselective retinoids. We examined the effect of LGD1069 (10 microm) on cellular proliferation and expression of putative intermediate biomarker genes including TGF-alpha, EGFR, and RAR-beta in seven SCCHN cell lines. A quantitative reverse transcription-PCR assay using a novel "primer dropping" method was used to determine expression levels of EGFR, TGF-alpha, and RAR-beta before and after treatment with LGD1069 (10 microM). SCCHN proliferation was reduced by a mean of 50% at 4 days in seven SCCHN cell lines after LGD1069 treatment (P < or = 0.05). EGFR expression levels were decreased by a mean of 58.4% (P = 0.007), TGF-alpha levels were decreased by a mean of 28.8% (P = 0.01), and RAR-beta levels were increased by a mean of 60% (P = 0.03). TGF-alpha stimulation of EGFR is associated with constitutive signal transducer and activator of transcription 3 (Stat3) activation in SCCHN. Abrogation of constitutive Stat3 activation was seen with LGD1069 treatment. These results suggest that an RXR-selective retinoic acid decreases SCCHN proliferation in part by interfering with TGF-alpha/EGFR autocrine signaling.

Retinoid-induced suppression of squamous cell differentiation in human oral squamous cell carcinoma xenografts (line 1483) in athymic nude mice.

Retinoids are promising agents for therapy of squamous cancers. In vitro, retinoids decrease expression of differentiation markers in head and neck squamous carcinoma cells. Little information is available on effects of retinoids on head and neck squamous carcinoma cell xenograft growth in vivo. To address this issue, head and neck squamous carcinoma cells (line 1483) were established as xenografts in nude mice. Control tumors grew rapidly with doubling times of 4-6 days to mean volumes of 1696 mm3 after 24 days. Histological analyses indicated the formation of well-differentiated squamous carcinoma cells exhibiting pronounced stratification (basal and suprabasal cells) and keratinization (keratin pearls) with abundant stroma. Cytokeratin 19 expression was restricted to the basal cell layers, and cytokeratin 4 expression was abundant in suprabasal cells. Mice were treated daily with 30 mg/kg 9-cis retinoic acid, 20 mg/kg all-trans-retinoic acid, or 60 mg/kg 13-cis retinoic acid by p.o. gavage on a schedule of 5 days/week over 4 weeks. Low micromolar (1.48-3.67 microM) and nanomolar (200-490 nM) concentrations of 9-cis retinoic acid and all-trans-retinoic acid were measured in plasmas and xenografts, respectively, 30 min after dosing. Retinoid treatment produced a marked suppression of the squamous cell differentiation of tumor cells manifest by decreased keratinization, loss of stratification, and accumulation of basal cells. This was accompanied by large decreases in the number of CK4-positive cells and concomitant increases of CK19-positive cells. REtinoic acid receptor-beta expression was also increased by 2.9-9.7-fold after chronic retinoid treatment. 9-cis retinoic acid and all-trans-retinoic acid decreased tumor volumes by 23 +/- 5 (SE) and 19 +/- 3%, respectively (P < or = 0.05); 13-cis retinoic acid was inactive. These retinoids did not decrease the rate of exponential tumor growth but increased the latent period until exponential growth began. These studies demonstrate that retinoids do not universally decrease tumor growth but profoundly suppress squamous cell differentiation in vivo in this xenograft model.

Differential effects of synthetic nuclear retinoid receptor-selective retinoids on the growth of human non-small cell lung carcinoma cells.

Retinoids are promising agents for cancer chemoprevention and therapy. Nuclear retinoic acid receptors (RARs; RARalpha, -beta, and -gamma) and retinoid X receptors (RXRs; RXRalpha, -beta, and -gamma) are thought to mediate most of retinoids' effects on cell growth and differentiation. Because the majority of human non-small cell lung carcinoma (NSCLC) cell lines are resistant to all-trans-retinoic acid, we searched for more potent retinoids. Therefore, we examined the effects of 37 natural and synthetic retinoids that exhibit specific binding to and transactivation of individual RARs or RXRs on the proliferation of eight human NSCLC cell lines. All of these cells expressed mRNAs of the three RXRs; however, they expressed varying levels of RARalpha and RARgamma, and only three of the eight cell lines expressed RARbeta mRNA. Cellular retinoic acid-binding proteins (CRABPs) I and II were detected in one and three of the eight cell lines, respectively. Only 8 of the 37 retinoids exhibited growth-inhibitory activity (IC50, < 10 microM) against at least two of the eight NSCLC cell lines. The active retinoids included one (TD550) of five RARalpha-selective, one (Ch55) of three RARbeta-selective, three (CD437, CD2325, and SR11364) of six RARgamma-selective, and one (CD271) of four RARbeta/gamma-selective retinoids. The potency of these retinoids was low (IC50, > 1 microM), except for CD437, which was very potent (IC50, 0.1-0.5 microM). The six RXR-selective retinoids were mostly inactive even at 10 microM. However, combinations of RAR-selective and RXR-selective retinoids exhibited additive effects. There appeared to be no simple correlation among the histological type of the NSCLC (adeno- or squamous), the levels of nuclear receptors or CRABPs, and the response of the cells to the growth-inhibitory effects of retinoids. Nevertheless, in contrast with former studies with natural retinoids, these results suggest that several synthetic retinoids do exhibit inhibitory activity against NSCLC cells, and some of them may be useful clinically.

Enhanced antitumor efficacy of cisplatin in combination with ALRT1057 (9-cis retinoic acid) in human oral squamous carcinoma xenografts in nude mice.

Cisplatin (DDP) is commonly used to treat head and neck tumors. Therapy frequently fails due to development of DDP resistance or toxicities associated with DDP therapy. In this study, effects of ALRT1057 [9-cis retinoic acid (9-cis RA)] on DDP cytotoxicity were studied in a human oral squamous carcinoma xenograft model. Mice bearing xenografts were dosed p.o. daily 5 days/week with 30 mg/kg 9-cis RA and/or i.p. twice weekly with 0.3-0.9 mg/kg DDP. Maximum tolerated doses of 9-cis RA and DDP were approximately 60 and >/=2.9 mg/kg, respectively, under their dosing schedules and routes of administration. Control tumors grew rapidly with mean doubling times of 4 +/- 1 days and reached mean volumes of 1982 +/- 199 (SE) mm3 after 24 days. DDP at doses of 0.3, 0.45, and 0.9 mg/kg inhibited tumor growth by 28, 47, and 86%, respectively, 24 days after tumor cell implantation. Thirty mg/kg 9-cis RA inhibited tumor growth by 25%. In combination, 0.3 mg/kg DDP + 30 mg/kg 9-cis RA inhibited tumor growth by 68%; 0.45 mg/kg DDP + 30 mg/kg 9-cis RA inhibited growth by 78%. These decreases were greater than those that would have been produced by either agent summed separately. Of importance, at doses of 9-cis RA that enhanced DDP cytotoxicity, no change in dose tolerance was observed as compared to tolerances observed for either agent alone, indicating that 9-cis RA increased sensitivity to DDP without altering systemic toxicity. In addition, 9-cis RA profoundly altered squamous cell carcinoma phenotypes by suppressing squamous cell differentiation, resulting in tumors with increased numbers of basal cells. In contrast, DDP selectively depleted proliferating basal cells from carcinomas. In combination, morphological changes produced by 9-cis RA alone predominated, suggesting a possible basis for enhanced DDP sensitivity in tumors exposed to both agents. These data demonstrate that 9-cis RA enhances tumor sensitivity to DDP, and suggest that this combination should be tested in Phase I-II clinical trials for its potential for improving anticancer therapy of squamous cell cancers.

Identification of receptor-selective retinoids that are potent inhibitors of the growth of human head and neck squamous cell carcinoma cells.

Retinoids modulate the growth and differentiation of cancer cells presumably by activating gene transcription via the nuclear retinoic acid receptor (RAR) alpha, beta, and gamma and retinoid X receptor (RXR) alpha, beta, and gamma. We analyzed the effects of 38 RAR-selective and RXR-selective retinoids on the proliferation of 10 human head and neck squamous cell carcinoma (HNSCC) cell lines. All of these cell lines expressed constitutively all of the receptor subtypes except RARbeta, which was detected in only two of them. Most of the RAR-selective retinoids inhibited the growth of HNSCC cells to varying degrees, whereas the RXR-selective retinoids showed very weak or no inhibitory effects. Three RAR antagonists suppressed growth inhibition by RAR-selective agonists, as well as by RAR/RXR panagonists such as 9-cis-retinoic acid. Combinations of RXR-selective and RAR-selective retinoids exhibited additive growth-inhibitory effects. Furthermore, we found that CD437, the most potent growth-inhibitory retinoid induced apoptosis and up-regulated the expression of several apoptosis-related genes in HNSCC cells. These results indicate that: (a) retinoid receptors are involved in the growth-inhibitory effects of retinoids; (b) RXR-RAR heterodimers rather than RXR-RXR homodimer are the major mediators of growth inhibition by retinoids in HNSCC cells; and (c) induction of apoptosis can account for one mechanism by which retinoids such as CD437 inhibit the growth of HNSCC cells. Finally, these studies identified several synthetic retinoids, which are much more effective than the natural RAs and can be good candidates for chemoprevention and therapy of head and neck cancers.

9-cis-Retinoic acid suppresses mammary tumorigenesis in C3(1)-simian virus 40 T antigen-transgenic mice.

Retinoids have been investigated as potential agents for the prevention and treatment of human cancers. These compounds play an important role in regulating cell growth, differentiation, and apoptosis. 9-cis-Retinoic acid (9cRA) is a naturally occurring ligand with a high affinity for both the retinoic acid receptors and the retinoid X receptors. We hypothesized that treatment with 9cRA would prevent mammary tumorigenesis in transgenic mice that spontaneously develop mammary tumors. To test this hypothesis, C3(1)-SV40 T antigen (Tag) mice, which develop mammary tumors by the age of 6 months, were treated daily p.o. with vehicle or two different dose levels of 9cRA (10 or 50 mg/kg) from 5 weeks to 6 months of age. Tumor size and number were measured twice each week, and histological samples of normal and malignant tissue were obtained from each mouse at time of sacrifice. Our results demonstrate that 9cRA suppresses mammary tumorigenesis in C3(1)-SV40 Tag-transgenic mice. Time to tumor development was significantly delayed in treated mice; median time to tumor formation for vehicle-treated mice was 140 days versus 167 days for mice treated with 50 mg/kg 9cRA (P = 0.05). In addition, the number of tumors per mouse was reduced by >50% in mice treated with 9cRA (3.43 for vehicle, 2.33 for 10 mg/kg 9cRA, and 1.13 for 50 mg/kg 9cRA, P < or = 0.002). Histological analysis of the mammary glands from vehicle and treated mice demonstrated that 9cRA treatment also did not affect normal mammary gland development. Immunohistochemical staining of normal and malignant breast tissue and Western blot analysis demonstrated that SV40 Tag expression was not affected by treatment with retinoids. Single doses of 10 and 50 mg/kg resulted in peak plasma concentrations of 3.4 and 6.71 microM, respectively. Daily doses of 9cRA for 28 days resulted in plasma concentrations of 0.86 and 1.68 microM, respectively, concentrations consistent with that seen in humans treated with 9cRA in clinical trials. These results demonstrate that 9cRA suppresses mammary carcinogenesis in transgenic mice without any major toxicity and suggest that retinoids are promising agents for the prevention of human breast cancer.

Discovery of novel retinoic acid receptor agonists having potent antiproliferative activity in cervical cancer cells.

Retinoic acid receptor (RAR) active retinoids have proven therapeutically useful for treating certain cancers and dermatological diseases. Herein, we describe the discovery of two new RAR active trienoic acid retinoids, (2E,4E,6E)-7-(3,5-di-tert-butylphenyl)-3-methylocta-2, 4,6-trienoic acid (10a, ALRT1550) and (2E,4E,6Z)-7-(3,5-di-tert-butylphenyl)-3-methylocta-2, 4,6-trienoic acid (10b, LG100567). ALRT1550 is a RAR selective retinoid which exhibits exceptional potency in both competitive binding and cotransfection assays. Moreover, it is the most potent antiproliferative retinoid described to date and thus has implications for the treatment of certain cancers. LG100567 is a potent panagonist which activates both RARs and retinoid X receptors.

A selective retinoid X receptor agonist bexarotene (LGD1069, targretin) inhibits angiogenesis and metastasis in solid tumours.

The present study determined the influence of a retinoid X receptor agonist bexarotene on angiogenesis and metastasis in solid tumours. In the experimental lung metastasis xenograft models, treatment with bexarotene inhibited the development of the lung tumour nodule formation compared to control. In vivo angiogenesis assay utilising gelfoam sponges, bexarotene reduced angiogenesis in sponges containing vascular endothelial growth factor, epidermal growth factor and basic fibroblast growth factor to various extent. To determine the basis of these observations, human breast and non-small-cell lung cancer cells were subjected to migration and invasion assays in the presence of bexarotene. Our data showed that bexarotene decrease migration and invasiveness of tumour cells in a dose-dependent manner. Furthermore, bexarotene inhibited angiogenesis by directly inhibiting human umbilical vein endothelial cell growth and indirectly inhibiting tumour cell-mediated migration of human umbilical vein endothelial cells through Matrigel matrix. Analysis of tumour-conditioned medium indicated that bexarotene decreased the secretion of angiogenic factors and matrix metalloproteinases and increased the tissue inhibitor of matrix metalloproteinases. The ability of bexarotene to inhibit angiogenesis and metastasis was dependent on activation of its heterodimerisation partner peroxisome proliferator-activated receptor gamma. Collectively, our results suggest a role of bexarotene in treatment of angiogenesis and metastasis in solid tumours.

Different genes control the susceptibility of mice to Moloney or Abelson murine leukemia viruses.

The susceptibility of mice to lymphoma induction by Moloney or Abelson murine leukemia virus has been compared in BALB/c, C57BL/6, and BALB/cXC57BL/6 recombinant inbred strains. BALB/c mice were found to be susceptible to lymphoma induction by either virus, and C57BL/6 mice were found to be relatively resistant to lymphoma induction by either virus. The genes that control these patterns of susceptibility to each virus are not the same because susceptibility to each virus segregated independently in CXB recombinant inbred strains. We also found, as reported by Cook (W. Cook, Proc. Natl. Acad. Sci. U.S.A. 79:2917-2921, 1982), when injected intrathymically that Abelson murine leukemia virus rapidly induced thymomas in weanling B6 mice. Examination of the cellular phenotypes of the tumors induced by Abelson murine leukemia virus or by Moloney murine leukemia virus indicated that different lymphocyte subpopulations were the targets for tumor induction by each virus.