Gerontology & Geriatrics Studies Homozygous Long-Lived Ames Dwarf Mice Isao Eto* University of Alabama at Birmingham, USA *Corresponding author: Isao Eto, University of Alabama at Birmingham, USA Submission: November 16, 2021 Published: December 03, 2021 DOI: 10.31031/GGS.2021.07.000663 ISSN 2578-0093Volume7 Issue3

Introduction. The molecular biological mechanism of the increased incidence of the various types of cancer in obesity or type 2 diabetes in rodents or humans has largely been resolved in recent years. By contrast, the molecular biological mechanism of the decreased, not increased, incidence of the various types of cancer in the homozygous long-lived Ames dwarf mice still remains unresolved. Objective. The first objective of the present study was to investigate whether the decrease in the incidence of cancer in the homozygous long-lived Ames dwarf mice is due to the increase, not decrease, in the expression of p27Kip1, a cell cycle repressor protein. The second objective was to investigate whether the decrease in the incidence of cancer in the homozygous long-lived Ames dwarf mice is due to the decrease, not increase, in the levels of glucose or insulin. Methods. To achieve these objectives, we first performed western immunoblot analysis of the hepatic expression of p27Kip1 protein. We then performed, using a human breast cancer cell line in vitro, the luciferase reporter plasmid assay to determine whether the translation initiation activity of the p27Kip1 mRNA is increased when the concentrations of either glucose or insulin are decreased. Results and Conclusion. The results of the first objective indicated that the hepatic expression of p27Kip1 protein was up-regulated in the homozygous long-lived Ames dwarf mice as expected. We also found that the lower concentrations of glucose or insulin increased the translation initiation activity of the p27Kip1 mRNA.

Research Aims: Obesity and type 2 diabetes are known to be associated with increased risk of various types of cancer. However, the molecular biological mechanism of how the risk of cancer is increased in obesity or type 2 diabetes is not known. The aim this research is to investigate if the decreased expression of p27Kip1, a cell cycle repressor protein, plays a central role in this mechanism. Research Methods, Previous Studies and Theoretical Backgrounds: It is well known that the expression of p27Kip1 is increased by numerous nutritional or chemopreventive anti-cancer agents. But it has never been known that the expression of p27Kip1 could be decreased, rather than increased, and the risk of cancer could be increased, rather than decreased. This problem was solved recently and this new analytical method was used in this study. Results: 1) The expression of p27Kip1 was indeed significantly decreased in human obese type 2 diabetic individuals relative to the lean normal controls. 2) The expression of p27Kip1 was also significantly decreased in genetically obese rodents relative to the lean normal controls. Additionally, in obese rodents, the concentrations of glucose or insulin were significantly increased relative to the lean normal controls. 3) Using human cells cultured in vitro it was found that the increased concentrations of glucose or insulin decrease the expression of p27Kip1. Conclusions: These results suggest that higher concentrations of glucose or insulin increase the risk of various types of cancer in obesity or type 2 diabetes by decreasing the expression of p27Kip1.

BackgroundBexarotene (Targretin®) is currently the only FDA approved retinoid X receptor (RXR) –selective agonist for the treatment of cutaneous T-cell lymphomas (CTCLs). The main side effects of bexarotene are hypothyroidism and elevation of serum triglycerides (TGs). The novel RXR ligand, 9-cis UAB30 (UAB30) does not elevate serum TGs or induce hypothyroidism in normal subjects.ObjectivesTo assess preclinical efficacy and mechanism of action of UAB30 in the treatment of CTCLs and compare its action with bexarotene.MethodsWith patient-derived CTCL cell lines, we evaluated UAB30 function in regulating growth, apoptosis, cell cycle check points, and cell cycle-related markers.ResultsCompared to bexarotene, UAB30 had lower half maximal inhibitory concentration (IC50) values and was more effective in inhibiting the G1 cell cycle checkpoint. Both rexinoids increased the stability of the cell cycle inhibitor, p27kip1 protein, in part, through targeting components involved in the ubiquitination–proteasome system: 1) decreasing SKP2, a F-box protein that binds and targets p27kip1 for degradation by 26S proteasome and 2) suppressing 20S proteasome activity (cell line-dependent) through downregulation of PSMA7, a component of the 20S proteolytic complex in 26S proteasome.ConclusionsUAB30 and bexarotene induce both early cell apoptosis and suppress cell proliferation. Inhibition of the G1 to S cell cycle transition by rexinoids is mediated, in part, through downregulation of SKP2 and/or 20S proteasome activity, leading to increased p27kip1 protein stability. Because UAB30 has minimal effect in elevating serum TGs and inducing hypothyroidism, it is potentially a better alternative to bexarotene for the treatment of CTCLs.

The p27Kip1 is a cell cycle repressor protein that regulates primarily the cell cycle transition from G1 to S phase and hence the DNA replication is in the S phase and cell division in the M phase. Expression of p27Kip1 protein has dual roles for both cancer prevention and promotion. For example, numerous nutritional and chemopreventive anti-cancer agents specifically increase the expression of p27Kip1 protein without directly affecting the expression of any other cell cycle regulatory proteins. On the other hand, pro-cancer agents (like glucose, insulin and other growth factors frequently seen in obesity and/or diabetes) specifically decrease the expression of p27Kip1 protein without directly affecting the expression of any other cell cycle regulatory proteins. Unlike expression of any other cell cycle regulatory proteins, expression of p27Kip1 protein is very unusual. The mRNA of p27Kip1 has a very long and unusual 5’-untranslated region (from -575 to -1 in human). It appears that the 5’-untranslated region of p27Kip1 mRNA forms two alternative secondary structures. One increases the expression of p27Kip1 protein when anti-cancer agents are added and another decrease the expression of p27K1p1 when pro-cancer agents are added. For this short concept proposal, Dr. Albert Einstein’s “visualized thought experiments (German: Gedanken experiment)” were used as a fundamental tool for understanding how either anti- or pro-cancer agents bring the primary structure of the 5’-untranslated region of p27Kip1 mRNA into two alternative secondary structures, thereby either increasing or decreasing, respectively, the translation initiation of p27Kip1 protein.

Bexarotene/Targretin®, a RXR selective ligand (rexinoid), is a commonly prescribed treatment for cutaneous T-cell lymphoma (CTCL). Long-term treatment with Targretin, however, elevates serum triglyceride (TG), which can lead to pancreatitis and cardiovascular disease. Thus, improved treatment for CTCL is needed. A potential candidate is a novel rexinoid, which does not elevate serum TG in animals and humans. The purpose of this study is to assess the efficacy and molecular mechanism of action of this rexinoid for the treatment of CTCL and compare its action with Targretin using human-derived CTCL cell lines, MyLa and HuT78, which are representative of Mycosis Fungoides (MF) and Sézary Syndrome (SS), respectively. Cell proliferation, cell cycle analyses and cell-cycle related markers were determined by hemocytometer, Flow cytometry analysis of propidium iodide-labeled cells and Western blot analysis, respectively. The rexinoid is as effective as Targretin in suppressing cell proliferation and arresting cell cycle at the G1 checkpoint in both cell lines. Furthermore, both drugs significantly (p < 0.05 relative to DMSO control) suppress the expression of Skp2 as determined by qRT-PCR and Western blot. The decrease in Skp2 protein resulted in a concomitant up-regulation of p27kip1 protein, the major inhibitor of cell cycle. These findings indicate that the novel rexinoid is effective in inhibiting G1 cell cycle checkpoints of CTCL cells, in part, by suppressing Skp2 expression resulting in increasing p27kip1 protein. In sum, the rexinoid with minimal effect in elevating serum TG, is an possible alternative to Targretin for the treatment of CTCL.

Introduction: The consensus report issued jointly by the American Diabetes Association and the American Cancer Society stated that "type 2 diabetes and cancer share many risk factors, but potential biologic links between the two diseases are incompletely understood". Interestingly, however, a recent report suggested that the expression of p27(Kip1), a cell cycle repressor protein, in the rodent liver was inversely associated with potential carcinogenic risk in the genetic rodent models of diabetic obesity. p27 is a cyclin-dependent kinase inhibitor that, when down-regulated, allows the progression of the cell cycle from G1 to S phase, thereby increasing the risk of developing cancer. Objective: The objective of the study described below was to extend the results of the recent report on the expression of p27 in the livers of obese, diabetic rodents to the humans and investigate whether the expression of p27 in the human peripheral blood mononuclear cells (PBMCs) might also be inversely associated with potential carcinogenic risk in obese type 2 diabetic individuals relative to the lean normal controls. Methods: Western immunoblot analysis was performed to evaluate the expression of p27 and the two most relevant upstream molecular signaling pathways of the expression of p27, namely 4E-BP1 and MNK1, in human PBMCs obtained from obese type 2 diabetic individuals relative to the lean normal controls. Results: First, expression of p27 in human PBMCs was significantly down-regulated in obese type 2 diabetic individuals relative to the lean normal controls. Secondly, expression of p27 in human PBMCs was also significantly down-regulated in obese type 2 diabetic African Americans relative even to the obese type 2 diabetic Caucasian Americans. Conclusions: Expression of p27 in human PBMCs was inversely associated with potential carcinogenic risk in obese type 2 diabetes relative to the lean normal controls.

Introduction The association of genetic rodent models of obesity and cancer still remains a controversial issue. Although this controversy has largely been resolved in recent years for homozygous leptin receptor-deficient obese Zucker rats and homozygous long-lived Ames dwarf mice, it is still unresolved for homozygous leptin-deficient obese ob/ob mice. Objective The objective of the present study described below was to investigate whether the expression of the cell cycle repressor protein p27(Kip1) is (a) down-regulated in the tumor-free homozygous leptin receptor-deficient obese Zucker rats as well as tumor-free homozygous leptin-deficient obese ob/ob mice and (b) up-regulated in the tumor-free homozygous long-lived Ames dwarf mice. Methods To achieve this objective, we first performed western immunoblot analysis of the hepatic expression of p27. We then performed western immunoblot analysis and proteomic analysis of the hepatic expression of the proteins involved in the upstream molecular signaling pathways for the expression of p27. Lastly, we analyzed the serum levels of glucose, insulin, and branched-chain amino acids, all of which have been shown to regulate, causally and inversely, the expression of p27. Results/Conclusions The results indicated that the hepatic expression of p27 was down-regulated in the homozygous leptin receptor-deficient obese Zucker rats and up-regulated in the homozygous long-lived Ames dwarf mice as expected. We also found that the hepatic expression of p27 was down-regulated in the homozygous leptin-deficient obese ob/ob mice. This last observation was not completely consistent with all of the results of the published studies where homozygous leptin-deficient obese ob/ob mice were used.

Background The objective of this study was to investigate whether the levels of glucose or certain amino acids could regulate the expression of a cell cycle repressor protein p27(Kip1), thereby dictating the risk of cancer in either obesity or caloric/dietary restriction. Previously, we identified and reported four different upstream molecular signaling pathways of p27 expression in human breast cancer cells. We called these four pathways as pathway #1, #2, #3 and #4. We found that 4-hydroxytamoxifen - but not tamoxifen - up-regulated the expression of p27 using pathway #1 which consisted mainly of receptor tyrosine kinases and mTORC1. We now investigate, using 4-hydroxytamoxifen as a reference anti-cancer agents, whether (a) the moderate increase in the concentration of D -(+)-glucose could down-regulate and, conversely, (b) the deficiency of D -(+)-glucose or certain L -amino acids could up-regulate the expression of p27 in these cells using pathway #2 which consists mainly of AMPK and mTORC1. Results Using human MDA-MB-231 breast cancer cells in vitro , these hypotheses were tested experimentally by performing p27-luciferase reporter transfection assays and western immunoblot analyses. The results obtained are consistent with these hypotheses. Furthermore, the results indicated that, although 4-hydroxytamoxifen used primarily pathway #1 to down-regulate the phosphorylation of 4E-BP1 and up-regulate the expression of p27, it also secondarily down-regulated the phosphorylation of S6K1. In contrast, the deficiency of D -(+)-glucose or L -leucine used primarily pathway #2 to down-regulate the phosphorylation of S6K1, but they also secondarily down-regulated the phosphorylation of 4E-BP1 and up-regulated the expression of p27. Finally, deficiency of D -(+)-glucose or L -leucine - but not 4-hydroxytamoxifen - up-regulated the expression of mitochondrial ATP5A and SIRT3. Conclusions (a) 4-Hydroxitamoxifen used primarily pathway #1 to up-regulate the expression of p27. (b) Moderate increase in the concentration of D -(+)-glucose used primarily pathway #2 to down-regulate the expression of p27. (c) Deficiency of D -(+)-glucose or L -leucine also used primarily pathway #2 to up-regulate the expression of p27. (d) Deficiency of D -(+)-glucose or L -leucine - but not 4-hydroxytamoxifen - up-regulated the expression of mitochondrial ATP5A in the Complex V of respiratory oxidation-phosphorylation chain and mitochondrial SIRT3. The SIRT3 is one of the seven mammalian anti-aging as well as anti-metabolic sirtuins.

BACKGROUND:p27(Kip1) is a cyclin-dependent kinase inhibitor that inhibits G1-to-S phase transition of the cell cycle. It is known that a relatively large number of nutritional and chemopreventive anti-cancer agents specifically up-regulate expression of p27 without directly affecting the expression of other G1-to-S phase cell cycle regulatory proteins including p21(Cip1Waf1). However, the upstream molecular signaling pathways of how these agents up-regulate the expression of p27 have not been well characterized. The objective of this study was to identify such pathways in human breast cancer cells in vitro using 4-hydroxytamoxifen, dexamethasone, and various retinoic acids as examples of such anti-cancer agents.RESULTS:Experimental evidence presented in the first half of this report was obtained by transfecting human breast cancer cells in vitro with proximal upstream region of p27 gene-luciferase reporter plasmids. 1) The evidence indicated that 4-hydroxytamoxifen, dexamethasone, and various retinoic acids up-regulated expression of p27 in both estrogen receptor-positive and negative human breast cancer cells in vitro. 2) The degree of up-regulation of p27 expression by these anti-cancer agents in human breast cancer cells in vitro linearly correlated with the degree of inhibition of methylnitrosourea (MNU)-induced rat mammary adenocarcinoma in vivo. 3) Lastly, up-regulation of the expression of p27 was likely due to the activation of translation initiation rather than transcription of p27 gene. The experimental evidence presented in the second half of this report was obtained by a combination of Western immunoblot analysis and transfection analysis. It indicated that 4-hydroxytamoxifen and dexamethasone up-regulated expression of p27 by down-regulating phosphorylation of eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) at Ser65 and this phosphorylation was likely to be mediated by upstream receptor tyrosine kinases/phosphoinositide-3-kinase/Akt/5'-AMP-activated protein kinase/mammalian target of rapamycin (RTKs/PI3K/Akt/AMPK/mTOR) protein kinase signaling pathways. Retinoic acids up-regulated expression of p27 without using either 4E-BP1 or RTKs/PI3K/Akt/AMPK/mTOR protein kinase signaling pathways.CONCLUSIONS:4-Hydroxytamoxifen and dexamethasone up-regulated translation initiation of p27 by down-regulating 4E-BP1 phosphorylated at Ser65 and this down-regulation seemed to be mediated by upstream RTKs/PI3K/Akt/AMPK/mTOR protein kinase signaling pathways. Retinoic acids also up-regulated translation initiation of p27, but without using any of these pathways.

This study evaluates the influence of the contamination of various metal compounds on a runaway reaction hazard of aqueous hydrogen peroxide. The test apparatus was assembled on an experimental basis. Especially, to avoid any catalytic effects, we used a glass vessel. Within the experiments conducted, iron, copper, chlorine ions accelerated the runaway reaction. However, nickel, potassium, sulfate and nitrate ions showed inert. In the case of iron and copper, there is a linear correlation between the reciprocal of additive's concentration and the logarithm of times to maximum temperature like Arrhenius equation, although the reason cannot be explained at the present. The important factors affected on the runaway reaction should be multivalency. The reactivity difference in several metal compounds is well explained from the factor.

Abstract Abstract #5035 Background: The U.S. National Cancer Institute currently lists obesity as one of the leading factors that increase the risk of developing breast cancer. Also it has been known since 1909 that, in rodents, caloric/dietary restriction is a potent and highly reproducible way to prevent various types of cancer, including mammary adenocarcinoma. However, the molecular mechanisms of how obesity or caloric/dietary restriction modulates the risk of developing breast cancer are not known. Objective: Objective of this study is to identify such molecular mechanisms. In our previous study, several nutrient sensing pathways that are highly likely to modulate the risk of developing breast cancer were identified using human breast cancer cell lines that were transfected with luciferase reporter construct of 5'-untranslated region of p27Kip1 gene. In the present study, we investigated the activity of these nutrient sensing pathways in the rodent models of obesity and hormonal dwarfism, a genetic model of caloric/dietary restriction. Methods: Three rodent models of obesity were used, namely (a) leptin receptor-deficient female obese Zucker rats, (b) leptin-deficient female ob/ob mice and (c) 60% fat diet-induced female obese mice. It was previously reported that the incidence of carcinogen-induced mammary adenocarcinoma is significantly higher in obese Zucker rats compared to the wild-type lean Zucker rats. For rodent model of hormonal dwarfism, we used long-lived female Ames dwarf mice. These mice are very similar to the mice subjected to caloric/dietary restriction; they are resistant to various type of cancer and live significantly longer than normal controls. The nutrient sensing pathways of p27Kip1 were investigated using three different methods, namely (a) blood metabolite analysis, (b) western immunoblot analysis, and (c) proteomic analysis. The livers were used for the latter two analyses because we were interested in investigating the nutrient sensing pathways upstream of p27Kip1. Results and Discussion: Obesogenic Nutrient Sensing Pathways of p27Kip1 – The obesogenic nutrient sensing pathways of p27Kip1 were detected in two rodent models of obesity, namely (a) leptin receptor-deficient obese Zucker rats and (b) leptin-deficient ob/ob mice. These pathways were not detected in the 60% fat diet-induced obese mice. One of the pathways consisted of (a) glucose → (b) AMP/ATP ratio → (c) 5'AMP-dependent kinase (AMPK) → (d) tuberous sclerosis complex (TSC) protein → (e) mammalian target of rapamycin (mTOR) → (f) translation initiation repressor protein 4E-BP1 → (g) p27Kip1→ (h) inceased risk of breast cancer. Another pathway consisted of (a) growth factors (including insulin) → (b) receptor tyrosine kinases (RTKs) → (c) phosphoinositide-3-kinase (PI3K) → (d) Akt/PKB → (e) TSC → (f) mTOR → (g) 4E-BP1 → (h) p27Kip1 → (i) increased risk of breast cancer. Leptogenic Nutrient Sensing Pathways of p27Kip1 – The leptogenic nutrient sensing pathways of p27Kip1 were detected in the long-lived Ames dwarf mice. These pathways were the same as those of obesogenic nutrient sensing pathways except that the amount, ratio or phosphorylation of the intermediates was reversed. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5035.

A DFT study of hydrogen peroxide (H2O2)-metal ion (M) complexes has been carried out, in order to explain a runaway reaction of hydrogen peroxide initiated by addition of metal ion. Basis sets considered are LANL2DZ for metal atom, and D95V, D95V+*, and 6-311+G* for H and O atoms. The B3LYP method is used. Hydrogen peroxide-metal ion (K+, Ni2+, Cu+, Cu2+, Fe2+, Fe3+) complexes (1:1), K+, Cu+, and Fe2+ complexes have been optimized However, no stable structures have been found for Ni2+, Cu2+ and Fe3+ complexes, because the excess charge transfer from the metal ion to H2O2 causes Coulomb repulsion. Multi-molecular complexes containing water molecules have been converged to stationary points, because the positive charges of metal atom properly disperse. The interaction of metal hydrated ions and hydrogen peroxide changed with charge distribution, hydrogen bond and coordination state of the complexes.

A214 Background: A marker for preclinical in vitro screening of potential nutritional and chemopreventive anti-breast cancer agents must be efficient and reliable. p27(Kip1) is a cyclin-dependent kinase inhibitor and, when up-regulated, p27 inhibits G1-to-S phase transition of the cell cycle, thereby inhibiting DNA replication and cell division. In this study, we investigated whether p27 could be a useful marker for preclinical in vitro screening of nutritional and chemopreventive anti-breast cancer agents. Methods: Human breast cancer (MCF7, MDA-MB-321, and AU565) and mouse epidermal (JB6) cells were transfected with luciferase reporter plasmid containing proximal 59-upstream region of p27 gene and then exposed to various nutritional and chemopreventive anti-breast cancer agents. Results: The results presented in the first half of this report show that various nutritional and chemopreventive anti-breast cancer agents fairly faithfully up-regulated synthesis of p27 in human breast cancer (MCF7, MDA-MB-321, AU565) and mouse epidermal (JB6) cells. Up-regulation appeared to be specific to p27 because synthesis of cyclin D1, E, and A, and p21Cip1/Waf1 was not modulated by these agents. Up-regulation of p27 is likely due to activation of translation rather than transcription of p27 because (a) up-regulation was mediated by the 59-untranslated region (-575) of the p27 gene and (b) the antibiotic actinomycin D, an inhibitor of transcription, did not attenuate the up-regulation of the synthesis of p27. The results, presented in the second half of this report, was obtained using the 59-untranslated region (-575) of p27 gene. The evidence suggested that cancer preventive agents up-regulate the synthesis of p27 by at least four different molecular signaling pathways: (a) Caloric restriction is likely to up-regulate the synthesis of p27 via 59-AMP-activated protein kinase (AMPK; a metabolic energy sensor or cellular fuel gauge), tuberous sclerosis complex (TSC), and mammalian target of rapamycin (mTOR). Amino acid deficiencies also up-regulate the synthesis of p27 using some components of this pathway. (b) 4-Hydroxytamoxifen (but not tamoxifen), genistein (but not genistin), daidzein, and probably other nutritional and chemopreventive anti-cancer agents up-regulate the synthesis of p27 via receptor protein tyrosine kinases (RPTKs), phosphoinositide 3-kinase (PI3K), phosphoinosite-dependent kinase (PDK), Akt/PKB and mTOR. (c) The synthesis of p27 is also up-regulated via RPTKs followed by MAPKs - MEK, ERK and p38MAPK - and probably MNK. Finally, (d) global hypomethylation of 59-m 7 G cap of mRNAs also up-regulates the synthesis of p27. Conclusion: Based on these findings, we conclude that p27 might be a marker of choice for preclinical in vitro screening of potential nutritional and chemopreventive anti-breast cancer agents.

BACKGROUND:p27(Kip1) is a cyclin-dependent kinase inhibitor. When up-regulated, p27 inhibits G1-to-S phase transition of the cell cycle. This report addresses the question of whether various nutritional and chemopreventive anti-cancer agents up-regulate the expression of p27 in preneoplastic and neoplastic cells.RESULTS:Experimental evidence presented in the first half of this report shows that these agents fairly faithfully up-regulate expression of p27 in mouse epidermal (JB6) and human breast cancer (MCF7, MDA-MB-321, and AU565) cells. Up-regulation appears to be specific to p27 because expression of cyclin D1, E, and A, and p21Cip1/Waf1 was not modulated by these agents. Up-regulation of the expression of p27 is likely due to the activation of translation rather than transcription of p27 because (a) up-regulation is mediated by the 5'-untranslated region (-575) of the p27 gene and (b) the antibiotic actinomycin D, an inhibitor of transcription, did not attenuate the up-regulation of p27. This latter finding is likely to preclude the existence of cryptic transcription factor binding site(s) in the 5'-untranslated region of p27 gene. The experimental evidence, presented in the second half of this report, was obtained using the 5'-untranslated region (-575) of p27 gene. The evidence suggests that cancer preventive agents up-regulate expression of p27 by at least four different molecular signaling pathways: (a) Caloric restriction is likely to up-regulate p27 expression via 5'-AMP-activated protein kinase (AMPK; a metabolic energy sensor or cellular fuel gauge), tuberous sclerosis complex (TSC), and mammalian target of rapamycin (mTOR). Amino acid deficiencies also up-regulate the expression of p27 using some components of this pathway. (b) 4-Hydroxytamoxifen (but not tamoxifen), genistein (but not genistin), daidzein, and probably other nutritional and chemopreventive anti-cancer agents could up-regulate expression of p27 via receptor protein tyrosine kinases (RPTKs), phosphoinositide 3-kinase (PI3K), phosphoinosite-dependent kinase (PDK), Akt/PKB and mTOR. (c) Expression of p27 could also be up-regulated via RPTKs followed by MAPKs--MEK, ERK and p38MAPK--and probably MNK. Finally, (d) global hypomethylation of 5'-m7G cap of mRNAs could also up-regulate expression of p27.CONCLUSION:Based on these findings, we conclude that various nutritional and chemopreventive anti-cancer agents up-regulate expression of p27 in (pre)neoplastic cells.

Various aspects of the chemopreventive and chemotherapeutic properties of the RXR receptor agonist Targretin (LGD 1069) were examined in the methylnitrosourea (MNU)-induced model of mammary cancer. The administration of Targretin at dose levels of 60, 20 or 6.7 mg/kg body wt/day by gavage decreased the number of mammary tumors by 96, 85 and 78%, respectively. When Targretin was administered in the diet at 92 and 275 mg/kg diet cancer multiplicities were reduced by 78 and 92%, respectively. A wider range of dietary doses of Targretin at 15, 50 and 150 mg/kg diet reduced the number of mammary tumors by 38, 55 and 70%, respectively. Treatment of rats with different regimens of Targretin (250 mg/kg diet) yielded cancer multiplicities of 4.3 for non-treated rats, 0.5 for rats treated continuously with Targretin, 2.1 for rats treated with Targretin for 8 weeks followed by 10 weeks of the control diet and 1.6 for rats treated with Targretin alternating 3 days on and 4 days off. Targretin was also examined as a therapeutic agent by treating rats with at least one palpable mammary tumor for 5 weeks. A high dose of Targretin (272 mg/kg diet) caused partial or complete regression of approximately 65% of the cancers over this time period. In contrast, in animals treated with 15 mg Targretin/kg diet only 1 of 12 cancers showed significant regression. Finally, the effect of a limited exposure to Targretin (7 days) on cell proliferation and apoptosis in small mammary tumors was determined. Targretin at 150 mg/kg diet strongly decreased proliferation (75%) and increased apoptosis (300%), while a lower dose of Targretin (15 mg/kg diet, which still prevented 30% of cancers) had no effect on apoptosis but did decrease cell proliferation. Determination of serum IGF1 levels showed that treatment of rats with highly effective doses of Targretin at 272 mg/kg diet or at 60 or 20 mg/kg body wt/day by gavage caused significantly decreased serum IGF1 levels.

3132 Treatment of rodents with OH-BBN results in the formation of a high percentage of urinary bladder cancers with mixed transitional cell and squamous cell components. In this study, rats were treated with OH-BBN (150 mg/gavage) twice a week for eight weeks. One week following the last dose of OH-BBN, animals were administered the chemopreventive agents either by gavage or diet containing the indicated dose levels for 27 weeks. Celecoxib was mixed directly in the diet, while rosiglitazone (in PEG 400: 0.5% carboxymethylcellulose; 50:50) and polyphenon E (in saline) were given daily (7x/week) by gavage. Rats administered OH-BBN only developed 20 large urinary bladder cancers among 35 treated rats. Interestingly, all 34 rats administered rosiglitazone (50 mg/Kg BW/day), a known PPAR gamma agonist, developed large palpable cancers resulting in early sacrifice of many of the rats. This suggests that rosiglitazone may be a tumor promoter in this model. Rats treated with polyphenon E (100 and 250 mg/Kg BW/day) yielded a dose dependent decrease in large tumors to 14/35 and 6/34 rats, respectively; implying significant chemopreventive activity. Rats treated with celecoxib in the diet (63, 250 or 1000 mg/kg diet) yielded a dose dependent decrease in the number of large tumors of 8, 4 and 2, respectively. When celecoxib (1000 mg/kg diet) was added beginning at 22 weeks following the first dose of OH-BBN and 14 weeks prior to the time of final sacrifice, the final number of tumors was still decreased from 20/35 (control diet) to 6/35 (late celecoxib). These studies show that celecoxib is effective over a wide range of doses, and even when given later during tumor progression. Further studies examining other histological lesions (e.g., hyperplasias, papillomas,and microscopic cancers) will be presented. (Supported by NCI-N01-CN-15128).