Homozygous long-lived Ames dwarf mice (males) (J181312q Prop1−/−) and wild-type normal Ames control mice (males) (J181312q Prop1+/+) were purchased initially from the Jackson Laboratory (Bar Harbor, MI, USA) at 9 to 10 weeks of age, but later they were kindly and generously provided by Dr. Andrzej Bartke at the Southern Illinois University (SIU) School of Medicine (Springfield, IL, USA).

At our institution (University of Alabama at Birmingham, Birmingham, Al., USA), all procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). All animals were housed in regular mouse cages on 12-h light/dark cycle (lights on at 7:00 am) and had unlimited access to food and tap water at our institution. All mice were fed Lab Diet ®5K52, which contained approximately 19% protein and included ground wheat, dehulled soybean meal, and fish meal. All animals were sacrificed at 12 weeks of age after overnight fast and the serum and liver samples were collected from each animal, snap-frozen in liquid nitrogen, and stored at −80˚C until processed further.

The following primary antibodies against p27Kip1 were purchased from the Cell Signaling Technology (Danvers, MA, USA) and the Santa Cruz Biotechnology (Santa Cruz, CA, USA). Additionally, primary antibodies against GAPDH were purchased from the Santa Cruz Biotechnology.

Western immunoblot analysis of the expression of p27Kip1 was performed using the livers of the mice. The soluble protein extracts of the livers were prepared by sonicating in RIPA Lysis Buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA) containing PMSF, sodium orthovanadate and protease inhibitor cocktail and supplemented with 50 mmol/L NaF. The soluble protein extracts were collected by centrifugation and stored at −80˚C until processed for western immunoblot analysis.

For western immunoblot analysis, the soluble protein extracts (35 to 50 μg protein per lane) were applied to the SDS-PAGE and, after fractionation, proteins were transferred to the nitrocellulose membrane, which was then blocked and incubated in a solution containing first primary antibody. After shaking overnight at 4˚C, the target proteins bound to the first primary antibody against p27Kip1 were further incubated in a solution containing alkaline phosphatase (AP)-conjugated secondary anti-immunoglobulin antibody and detected by chemiluminescence using TROPIX Western-Star Kit (Applied Biosystems, Foster City, CA, USA). After exposure to X-ray film, the blots were stripped using Western Re-Probe Solution (G-Biosciences, St. Louis, MO, USA), checked for removal of the chemiluminescence and then re-probed with second primary antibody against GAPDH.

Densitometric measurements of the intensity of the bands on the X-ray films were performed using UN-SCAN-IT Gel & Graph Digitizing Software (Silk Scientific, Orem, UT, USA). Background corrections were done by four corner interpolation method and optical density calculations were performed using linear standard reflective scan method.

Serum levels of glucose and insulin were determined at our laboratory (Department of Nutrition Sciences, University of Alabama at Birmingham (UAB)) (Birmingham, AL, USA). This laboratory has been jointly operated by the NIH-funded Pittman General Clinical Research Center and Nutrition Obesity Research Center at UAB.

Glucose was determined by glucose oxidase method. Insulin was determined by double-antibody radioimmunoassay procedure using RIA Linco kits manufactured by the Linco Research (St. Charles, MO, USA).

Human breast cancer cell line MDA-MB-231 in vitro was obtained from the American Type Culture Collection (Rockville, MD, USA). The cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 4.5 g/L of d-(+)-glucose, supplemented with 10% heat-inactivated FBS, 2% l-glutamine, and antibiotics. All cells were subcultured after trypsinization with 0.05% trypsin-0.02% EDTA solution. The incubation was carried out at 37˚C in a 5% CO₂ humidified chamber. The cultures were always maintained below confluency. The cells were checked periodically for mycoplasmal infection by DNA fluorochrome staining.

LY 294002, rapamycin, d-(+)-glucose, and rotenone were obtained from Sigma-Aldrich (St. Louis, MO, USA). Triciribine, AG1024, AGL2263, IGF-1R inhibitor PPP, compound C, and metformin were obtained from Calbiochem/EMD (San Diego, CA, USA). AICA riboside was purchased from Phoenix Pharmaceuticals, Inc. (Belmont, CA, USA). Dulbecco’s Modified Eagle’s Medium (DMEM) labeling kit was obtained from Chemicon International (Temecula, CA, USA).

Luciferase reporter plasmid containing from −575 to −1 untranslated region (p27Kip1-5’UTR) of the p27Kip1 mRNA [6] [7] in the proximal 5’-upstream region of the p27Kip1 gene was used to transfect the cells. The control luciferase reporter plasmid that does not contain the insert was also prepared to test if any unknown pro-cancer agents or nutritional/chemopreventive anti-cancer agents were exerting any spurious effects on the backbone, rather than the insert, of the luciferase reporter plasmid. We found that none of the agents tested exerted any spurious effects on the backbone of the luciferase reporter plasmid in the human breast cancer cells used.

Transfections were performed according to the published protocol [30] using FuGENE 6 purchased from the Roche Applied Science (Indianapolis, IN, USA). In brief, 24 hours before reporter transfection, the cells were seeded into a 60-mm tissue culture dish at a density of 1.5 × 10⁵ cells/dish and incubated at 37˚C in a 5% CO₂ humidified chamber. Reporter transfection was then carried out with 1 µg of luciferase reporter plasmid and 0.2 µg of pSV-β-galactosidase internal control plasmid (Promega, Madison, WI, USA) mixed with 3 µL of FuGENE 6 solution in 3 mL of FBS-free DMEM supplemented with only 2% l-glutamine. A minimum of 5-hour incubation at 37˚C was needed for transient transfection, followed by 18-hour incubation at 37˚C in a 5% CO₂ humidified chamber in normal DMEM with 10% FBS for recovery. The transfected cells were then starved in DMEM with 0.2% FBS at 37˚C in a 5% CO₂ humidified chamber for 24 hours.

The resulting cells were treated with either various compounds or vehicle controls in DMEM with 0.2% FBS at 37˚C in a 5% CO₂ humidified chamber for 24 hours. For studies with zero concentration of glucose, DMEM labeling kit was purchased from the Chemicon International (Temecula, CA, USA), but only glucose was omitted from the kit. The cells, transfected with luciferase reporter plasmid in normal DMEM with 10% FBS and starved in normal DMEM with 0.2% FBS for 24 hours as described above, were then incubated in either normal concentration (5.55 mM) or zero concentration of d-(+)-glucose in glucose-deficient DMEM with 0.2% FBS at 37˚C in a 5% CO₂ humidified chamber for 24 hours.

After 24 hours, the treated cells were collected and lysed using Reporter Lysis Buffer (Promega, Madison, WI). The resulting cell lysates were assayed for luciferase activity using Luciferase Assay Kit (Promega, Madison, WI, USA) and TD-20/20 Luminometer (Turner Designs, Sunnyvale, CA, USA). β-Galactosidase activity was measured using chlorophenol red-β-d-galactopyranoside (CPRG) (Sigma-Aldrich, St. Louis, MO, USA) as a substrate.

To assess the regulation of the hepatic expression of p27Kip1, a cell cycle repressor protein, the long-lived Ames dwarf mice and normal Ames control mice were used for this study (Figure 1(a)). All mice were obtained at 9 to 10 weeks of age and sacrificed at 12 weeks of age. These mice were all males because the levels of the expression of p27Kip1 were generally gender neutral based on our preliminary studies. These animals were also considered “tumor-free” based on the following two criteria: 1) they were generally tumor free at this younger age as confirmed by our resident pathologist and the commercial suppliers and also 2) they had not been treated with any known chemical carcinogens.

The body weights of the long-lived Ames dwarf mice were significantly lower—by about 63%—relative to the normal Ames control mice (Figure 1(b)). At 12 weeks of age, the average body weight of the long-lived Ames dwarf mice was about 10 grams and that of the normal Ames control mice was about 27 grams. The P-values in the difference in the body weights between long-lived Ames dwarf mice and normal Ames control mice were below 0.01. This may be due to the fact that the homozygous dwarf mice were not fertile and, therefore, dwarf gene must be maintained in the heterozygotes. So, it is likely that the homozygous long-lived Ames dwarf mice and the wild-type normal Ames control mice were siblings.

In contrast to the body weights, western immunoblot analyses of the levels of the hepatic expression of p27Kip1 were significantly higher—by about 74%—in the long-lived Ames dwarf mice relative to those of the normal Ames control mice (Figure 1(c)).

Figure 1(d) shows the overview of the molecular biological effects of the increasing expression of p27Kip1, a cell cycle repressor protein, on the cell cycle in the long-lived Ames dwarf mice relative to the normal Ames control mice. The results of the preliminary studies have indicated that the increased expressions of p27Kip1 decrease the risk of cancer by inhibiting the G1-to-S phase transition of the cell cycle, thereby decreasing the DNA replication in the S phase and cell division in the M phase. Numerous nutritional and chemopreventive anti-cancer agents decrease the risk of cancer by increasing the expression of p27Kip1. These effects of the increased expressions of p27Kip1 appear to be specific to the p27Kip1. No other cell cycle regulatory proteins are known to cause these effects.

The serum concentrations of glucose were significantly lower—by about 68%—relative to the normal Ames control mice (Figure 2(a)). At 12 weeks of age, the average concentrations of the serum glucose of the long-lived Ames dwarf mice were about 112 mg/dl and those of the normal Ames control mice were about 350 mg/dl. Similarly, the serum concentrations of insulin were also significantly lower—by about 78%—relative to the normal Ames control mice (Figure 2(b)). At 12 weeks of age, the average concentrations of the serum insulin of the long-lived Ames dwarf mice were about 0.125 ng/ml and those of the normal Ames control mice were about 0.58 ng/ml.

Figure 2(c) illustrates the tentative hypothesis of how the lower concentrations of serum glucose or insulin might inhibit the cell cycle by increasing the expression of p27Kip1, a cell cycle repressor protein, in the long-lived Ames dwarf mouse. This hypothesis is identical to that presented above in Figure 1(d) except that we now propose more specifically that the lower concentrations of serum glucose or insulin are the ones that might be responsible for increasing the expression of p27Kip1, a cell cycle repressor protein, thereby decreasing the risk of various types of cancer in the long-lived Ames dwarf mouse.

Molecular biological mechanism of the regulation of the expression of p27Kip1 protein is unusual. First of all, expression of p27Kip1 is regulated primarily at the level of translation, not at the level of transcription. Second, its mode of translation initiation is cap independent, not cap dependent. Thus, investigation of the potential regulation of the translation initiation activity of the p27Kip1 mRNA by lower concentrations of glucose or insulin must be performed carefully.

Our preliminary studies have indicated that the best approach to estimate the translation initiation activity of the p27Kip1 mRNA by glucose or insulin was to use the luciferase reporter plasmid that contains only a portion of the p27Kip1 gene that corresponds to the proximal 5’-upstream untranslated region (5’UTR) of the p27Kip1 mRNA. The primary RNA structure of this luciferase reporter plasmid is reproduced in Figure 3. It consists of the RNA bases from −575 to −1. A more comprehensive description of this portion of the p27Kip1 mRNA will be presented later in the Discussion section of this manuscript. We reported recently that the molecular downstream pathways of how higher concentrations of glucose or insulin decrease the translation initiation activity of p27Kip1 mRNA by transiently transfecting this luciferase reporter plasmid into the MDA-MB-231 human breast cancer cells in vitro [6] [7]. It made sense, therefore, to use the same approach to investigate the opposite situation of how lower concentrations of glucose or insulin decrease the risk of various types of cancer by increasing the translation initiation activity of p27Kip1 mRNA.

The results shown in Figure 4(a) indicate that the removal of d-(+)-glucose from the cell culture medium for 24 hours significantly up-regulated the translation initiation activity of p27Kip1 mRNA (p27Kip1-5’UTR) relative to the normal concentration (5.55 mM) of glucose. The significant up-regulation of the translation initiation activity of p27Kip1 mRNA was also observed in the lower concentration (5.55 mM) of glucose relative to that when higher concentration (20 mM) of glucose was added to the culture medium for 24 hours.

Figure 4(b) shows that those compounds, circled in orange color, increased the translation initiation activities of p27Kip1 mRNA in the MDA-MB-231 cells in vitro. These compounds included rotenone, AICA riboside, metformin, and rapamycin. The other compounds, circles in green color, decreased the translation initiation activities of p27Kip1 mRNA in the same cells in vitro. These compounds included compound C and one or more of the branched chain amino acids. The 4E-BP1 (eukaryotic translation initiation factor 4E-binding protein 1 (eIF4E-BP)), circled in red color, decreased the protection of 5'-end cap of the p27Kip1 mRNA (p27Kip1-5’UTR), thereby increasing the cap-independent translation initiation activity of the p27Kip1 mRNA.

Figure 5(a) shows that certain inhibitors of insulin receptor appeared to have significantly and specifically increased the translation initiation activity of p27Kip1 mRNA (p27Kip1-5’UTR) in these cells in vitro. Of the three inhibitors used, two of them—IGF-IR inhibitor PPP and AGL2263 (IR and IGF-1R inhibitor)—significantly and specifically increased the translation initiation activity of p27Kip1 mRNA (p27Kip1-5’UTR) in MDA-MB-231 cells in vitro, but AG1024 (IGF-1R inhibitor) failed to increase it. These results suggested that the lower concentrations of insulin increased the translation initiation activity of p27Kip1 mRNA in vitro.

Figure 5(b) shows that those compounds, circled in orange color, increased the translation initiation activities of p27Kip1 mRNA in the MDA-MB-231 cells in vitro. These compounds included 1) the inhibitor of the receptor tyrosine kinase for the platelet derived growth factor; 2) LY 294,002 (inhibitor of phosphoinositide 3-kinase); and 3) triciribine (inhibitor of Akt/PKB).

The downstream pathway of increasing the translational activity of p27Kip1 mRNA by lower concentrations of insulin merges with the downstream pathway of lowering the concentrations of d-(+)-glucose at the point of the tuberous sclerosis proteins, Subsequently, the 4E-BP1, that is circled in red color, acts in the same way as for d-(+)-glucose; the 4E-BP1 decreases the protection of 5'-end cap of the p27Kip1 mRNA (p27Kip1-5’UTR), thereby increasing the cap-independent translation initiation of the p27Kip1 mRNA.