76 �� 1 12 in group A, and 3 36 �� 1 60 in group B (P = 0 01) Th

76 �� 1.12 in group A, and 3.36 �� 1.60 in group B (P = 0.01). The tumor considering necrosis rate at 1 mo post-RFA was 90.67% (68/75 lesions) in group A and 90.20% (92/102 lesions) in group B. HCC recurrence rate at 6 mo post-RFA was 17.33% (13/75) in group A and 31.37% (32/102) in group B (P = 0.04). CONCLUSION: PAA blocked effectively the feeding artery of HCC. Combination of PAA and RFA significantly decreased post-RFA recurrence and provided an alternative treatment for hypervascular HCC. Keywords: Hepatocellular carcinoma, Feeding artery, Radiofrequency ablation, Recurrence, Color Doppler flow imaging INTRODUCTION Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world (564 000 cases per year) and the third most frequent cause of cancer-related death[1].

Surgical resection is considered to be potentially curative therapy. However, only about 20% of HCC patients are eligible for resection[1,2]; the remainder are ineligible because of multifocal tumors, advanced tumors, tumor location precluding complete resection, or poor hepatic functional reserve. Therefore, a variety of imaging-guided tumor ablation therapies such as ethanol injection, microwave coagulation, percutaneous radiofrequency ablation (RFA) and laser ablation are often considered as alternative options[3�C6]. Among them, RFA has been used increasingly as a safe technique for treating hepatic tumors[7�C9]. However, for hypervascular HCC, RFA appears less effective because of a blood-flow-induced heat sink effect, which might cause incomplete ablation or recurrence[10].

Transcatheter arterial chemoembolization (TACE) can reduce blood supply of HCC by occlusion of tumor arteries, and the efficacy Anacetrapib of combination TACE and RFA has been confirmed[11,12]. Treatment difficulty remains for those patients who cannot tolerate or are ineligible for TACE because of liver cirrhosis or difficulty in manipulation of vessels with abnormal curvature that have resulted from surgical resection and liver transplantation. We hypothesized that, if percutaneous ablation of the feeding artery (PAA) of HCC could block or reduce the blood flow of HCC, the ablation volume of coagulation necrosis of subsequent RFA of the tumor would be increased. To the best of our knowledge, the application of PAA in the treatment of hypervascular HCC has not been reported in a large number of patients. In the present study, we evaluated the feasibility and adjuvant value of PAA performed before routine RFA treatment (PAA-RFA) of hypervascular HCC. MATERIALS AND METHODS Patients From January 2003 to June 2007, patients with HCC who met the entry criteria and agreed to participate were included in the study.

Obviously, immunoprophylaxis of perinatal HBV infection, implemen

Obviously, immunoprophylaxis of perinatal HBV infection, implemented since 1986 on a national basis, has been insufficient to prevent horizontal HBV/A infection diffusing among high-risk groups by http://www.selleckchem.com/products/DAPT-GSI-IX.html transmission routes shared by HIV infection. The foreseeable spread of HBV/A infection in Japan should be prevented by universal vaccination programs extended to high-risk groups or the general population. Acknowledgments The study was supported in part by a grant-in-aid from the Ministry of Health, Labor and Welfare of Japan and a grant-in-aid from the Ministry of Education, Culture, Sports, Science and Technology. We thank T. Kimura and K. Sato, Institutes of Immunology Co., Ltd.

(Tokyo, Japan), for determining HBV genotypes in this study and Takashi Saito, Yamagata University Hospital; Akihiro Matsumoto, Shinshu University Hospital; Yasuhiro Asahina, Musashino Red Cross Hospital; Yoshito Ito, University Hospital, Kyoto Prefectural University of Medicine; Keiko Hosho, Tottori University Hospital; Morikazu Onji, Ehime University Hospital; Tatsuya Ide, Kurume University Hospital; and Hiroshi Sakugawa, Hospital, University of the Ryukyus, for their help throughout this work. Kentaro Matsuura wrote the study protocol and the first draft of the manuscript and performed the experiments and statistical analysis. Yasuhito Tanaka contributed to the experimental work and the final version of the manuscript.

Shuhei Hige, Gotaro Yamada, Yoshikazu Murawaki, Masafumi Komatsu, Tomoyuki Kuramitsu, Sumio Kawata, Eiji Tanaka, Namiki Izumi, Chiaki Okuse, Shinichi Kakumu, Takeshi Okanoue, Keisuke Hino, Yoichi Hiasa, Michio Sata, and Tatsuji Maeshiro contributed to the collection of the samples and clinical data from patients and to the final version of the manuscript. Fuminaka Sugauchi, Shunsuke Nojiri, Takashi Joh, and Yuzo Miyakawa contributed to the final version of the manuscript. Masashi Mizokami had the original idea and did the planning of the study and contributed to the final version of the manuscript. All of the authors have seen and approved the final draft of the manuscript. Footnotes Published ahead of print on 18 March 2009.
The antibiotics and iron chelators used in this study are listed in Table 1 and were obtained from Sigma (St. Louis, MO), with the exception of deferasirox, which was a generous gift from Novartis (Basel, Switzerland).

Stock solutions of compounds were prepared in minimum essential medium (MEM) and made fresh immediately before use. Unless otherwise stated, conalbumin, DFO, and DSX were used at 20 ��g/ml, 400 ��g/ml, and 1 ��M respectively. These concentrations are able to chelate the approximately 0.1 ��M iron present in the medium. In addition, the concentrations of DFO and DSX used in this study were lower than or Cilengitide equal to the daily doses used in clinical practice to treat chronic iron overload.

The Khatri population was chosen because of its relatively higher

The Khatri population was chosen because of its relatively higher prevalence of diabetes as compared to other Sikh castes. Khatri Sikhs are more affluent and live in cities and are traders by profession. In general, Sikhs do not smoke for religious and cultural reasons and about 50% of the study participants are life-long vegetarians. A total of 1,115 individuals selleck chem Wortmannin from 338 families were extensively phenotyped [13]. DNA samples of 870 individuals (526 male/344 female) comprising 685 T2D cases and 185 normal glucose tolerant (NGT) relatives were successfully genotyped and used in this investigation. The T2D cases were 25 years or older and mean age at the time of recruitment (mean �� standard deviation [SD]) was 54.2��11.0 years. Average age of unaffected relative was 46.0��14.

7 years with a minimum age of 19 years. Only individuals who reported that all four grandparents were Khatri Sikhs of North Indian origin, who had Khatri surnames, and who spoke the Punjabi language were included. In addition, probands were required to have two or more full siblings with diabetes, or at least one living parent, and more than two siblings available for sampling. Excluded from the sample were half-siblings, adopted individuals, and individuals of South, East and Central Indian origin; individuals with type 1 diabetes (T1D) or a family member with T1D; individuals with rare forms of T2D such as maturity-onset diabetes of young (MODYs), or secondary diabetes (e.g., due to hemochromatosis or pancreatitis). Clinical characteristics of the SDS participants used for this investigation are summarized in Table 1.

All blood samples were obtained at the baseline visit. All participants provided a written consent following an informed consent procedures approved by Institutional Review Boards (IRBs). All SDS protocols and consent documents were reviewed and approved by the University of Oklahoma Health Sciences Center (OUHSC) (IRB # 13302, approved till August 31, 2011) and the University of Pittsburgh (IRB # 021234) as well as the Human Subject Protection (Ethical) committees at the participating hospitals and institutes in India. The Ethical committees of local institutions in India were Hero DMC Heart Institute, Ludhiana, and Guru Nanak Dev University, Amritsar. Each Institute in India also separately obtained Federal Wide Assurance (FWA) from the Office of Human Research Protection (OHRP) from the US Department of Health and Human Services (DHHS).

All the key investigators and key personnel working for SDS obtained online training for Human Participant Protection Education for Research. Table 1 Characteristics of Study Population Stratified by Gender and Disease (Mean �� SD). SDS Families A total AV-951 of 557 families were investigated and 236 families were excluded because they did not meet the eligibility criteria for the study.

Although all animals used in the present study had normal insulin

Although all animals used in the present study had normal insulin and glucose levels, indicating possibly normal glucose disposal in liver, muscle, and visceral fat, two out of four contained large adipocytes that were insulin resistant and showed no correlation between cell sizes and www.selleckchem.com/products/MLN-2238.html FA uptake (Fig. 7 and Table 1). Recent studies have argued against differential insulin sensitivity of adipocytes from high-fat diet and insulin-resistant mice, since small and large cells had similar insulin sensitivity (53). We hypothesize that, under physiological conditions, insulin-sensitive lipid uptake (the present study) and the antilipolytic action of insulin in adipose tissue (19, 38) are negatively regulated by cell size.

This mechanism may protect adipose tissue from lipid overload and the development of complications associated with the enlargement of adipocyte size, such as local hypoxia, inflammation, elevated basal lipolysis, and systemic insulin resistance (46, 50, 53, 55). Consistent with this hypothesis, adipose tissue insulin receptor knockout (FIRKO) mice are protected against obesity and obesity-related glucose intolerance (6). These effects may be mediated by factors other than the impaired glucose transport in adipocytes, because mice with local inactivation of the GLUT4 transporter in adipose tissue develop muscle and liver insulin resistance and glucose intolerance (1). Similar to FIRKO mice, FATP1-knockout mice are also protected against diet-induced obesity and systemic insulin resistance, and insulin-stimulated FA uptake in adipocytes is completely abolished (52).

These and earlier findings support our study and the hypothesis that, as the lipid storage capacity of adipocytes reaches threshold values, a negative feedback mechanism begins to inhibit insulin-dependent lipogenic processes, restricting further lipid accumulation and increased cell size. This cell size-sensing mechanism is likely to control multiple aspects of adipocyte function, including lipid metabolism and insulin signaling, and is likely activated by changes in biophysical properties or lipid chemistry of fat cells (13, 26, 54). Exte
nadph oxidases are a major source of superoxide production in the vasculature that contributes to endothelial dysfunction and vascular cell proliferation (4, 19).

In nonphagocytic cells, the catalytic moiety of NADPH oxidases is composed of one or more gp91phox (Nox2) homologs, Nox1, -3, -4, or -5, Duox1, or Duox2 (27). These Nox homologs associate with the membrane-bound p22phox subunit to generate reactive oxygen species (ROS). Nox4 is highly expressed in vascular wall cells including Drug_discovery smooth muscle and endothelial cells (47). In contrast to the other Nox homologs, current evidence indicates that Nox4 is constitutively active (1), and increases in Nox4 mRNA levels increase Nox4 activity (45).

7%) and two in the 400 IU/kg group (33 4%) were vitamin D insuffi

7%) and two in the 400 IU/kg group (33.4%) were vitamin D insufficient. All the other animals could be considered to have adequate 25-D3 levels. Serum calcium concentrations remained relatively stable and did not correlate with serum selleck chem Crizotinib 25-D3 levels (data not shown). Systemic levels of the active 1,25-D3 are regulated by the kidneys; therefore, we measured gene expression of the vitamin D activating enzyme 25-hydroxyvitamin D3 1��-hydroxylase (cyp27b1), the vitamin D degrading enzyme 1,25-hydroxyvitamin D3 24-hydroxylase (cyp24a1), and the vitamin D receptor (vdr) by qRT-PCR. High dietary vitamin D intake increased cyp24a1 (��: 0.518, p = 0.005) and inhibited cyp27b1 (��: ?0.452, p = 0.016) gene expression. Furthermore, cyp27b1 expression correlated negatively with serum 25-D3 levels (��: ?0.

419, p = 0.026, Fig. 4, Table 2). The expression of vdr was not influenced either by diet or serum 25-D3 levels. Fig. 4 High dietary vitamin D increases cyp24a1, decreases cyp27b1 and does not affect vdr expression. Effect of increasing vitamin D concentration in the diet on renal cyp24a1 (A), cyp27b1 (B) and vdr (C) expression. Circles represent relative Discussion The aim of this study was to find the?expression of … 4. appropriate (lowest) dietary vitamin D level that would prevent formation of premalignant lesions in a mouse model of chemically induced colonic tumorigenesis. To our knowledge this is the first study investigating the effect of dose-dependency of vitamin D intake on preventing AOM/DSS-induced colonic tumorigenesis in mice.

We could show unequivocally that increasing vitamin D intake was able to delay formation of chemically induced low grade preneoplastic lesions and even prevent development of high grade dysplasia in the colon. We observed a very clear inverse correlation between dysplasia score and higher vitamin D intake as well as serum 25-D3 levels. Our data support the hypothesis that increasing vitamin D in the diet has chemopreventive effects on chemically induced precursors of colorectal tumors in mice. This effect seems to plateau at an intake of 2500 IU vitamin D/kg diet or higher. The effect of vitamin D, its metabolites and vitamin D analogues on colonic tumorigenesis in mice was investigated in several studies, however, the results are contradictory.

Majority of the studies that were not able to show any preventing or delaying effect of vitamin D and its analogues on colorectal tumorigenesis used vitamin D-sufficient control groups. In these studies, control diets contained 1000 IU/kg vitamin D [18,19], a level that is probably already chemopreventive. Our study, similar to the data presented by Fleet et al. [20], shows that Entinostat 1000 IU vitamin D/kg diet provides 25-D3 serum levels substantially higher than 30 ng/ml. Nevertheless there were reports for the effectiveness of vitamin D even compared with standard control diet, e.g. the study by Fichera et al.

Quantitative PCR primers: p16INK4a (F: 5��-CCCAACGCCCCGAACT-3��,

Quantitative PCR primers: p16INK4a (F: 5��-CCCAACGCCCCGAACT-3��, R: 5��-GTGAACGTTGCCCATCATCA-3��); Cdkn1a (F: 5��-TTGCACTCTGGTGTCTGAGC-3��, R: 5��-TCTGCGCTTGGAGTGATAGA-3��); sellckchem Rassf1 (F: 5��-GCACTCTTTGAGCGAACTGA-3��, R: 5��-AGCTCAGGGCTTTTTCACTG-3��); Bmi1 (F: 5��-TGTCCAGGTTCACAAAACCA-3��, R: 5��-TCATTTTTGAAAAGCCCTGG-3��); Ezh2 (F: 5��-TTACTGCTGGCACCGTCTGATGTG-3��, R: 5��-TGTCTGCTTCATCCTGAGAAATAATCTCC-3��); Eed (F: 5��-GCACAGAGATGAAGTTCTGAGTGCTG-3��, R: 5��-ATAAGACTCCTTAATTGCATTCATCATCCT-3��); Bad (F: 5��-AGAGTATGTTCCAGATCCCAG-3��, R: 5��-GTCCTCGAAAAGGGCTAAGC-3��); Bax (F: 5��-GCTCTGAACAGATCATGAAG-3��, R: 5��-GATGGTCACTGTCTGCCATG-3��); Bcl-2 (F: 5��-TTGTGGCCTTCTTTGAGTTCG-3��, R: 5��-ATTTCTACTGCTTTAGTGAACC-3��); c-fos (F: 5��-GGTCATCGGGGATCTTGC-3��, R: 5��-ATGGGCTCTCCTGTCAAC-3��); c-jun (F: 5��-GATGGAAACGACCTTCTACGAC-3��, R: 5��-ACGTTCTTGGGGCACAAGAACT-3��); and GAPDH (F: 5��-CCTGCTTCACCACCTTCTTGA-3��, R: 5��-TGTGTCCGTCGTGGATCTGA-3��).

RNA Fluorescence in Situ Hybridization For RNA-fluorescence in situ hybridization, a 562-bp RNA-fluorescence in situ hybridization probe for nascent p16INK4a transcripts was derived with the primers 5��-CGTTCACGTAGCAGCTCTTC-3�� and 5��-GCCTTCGCTCAGTTTCTCAT-3��, which were designed from Primer Premier 5 and Oligo 6. Then another PCR was performed by adding the T7 promoter primer to the sense primer and the SP6 primer to the antisense primer from the previous reaction. The PCR product was purified with the Mini-DNA fragment Rapid Purification Kit (BioDev), and 1 ��g of the product was labeled by in vitro transcription with a Biotin-dUTP RNA labeling kit (Roche Applied Science).

The RNA product from the SP6 promoter was used as the probe for p16INK4a, and that from T7 was used as Drug_discovery a control. The product was purified with Quick Spin RNA Columns (Qiagen). For each slide, 50 ng of DNA probe, 5 ��g of salmon sperm DNA, and 20 ��g of tRNA were used. Two volumes of 100% EtOH were added. After being dried, the samples were resuspended in 10 ��l of hybridization mix and 1 ��l of RNA guard and left to dissolve at 37 ��C and then denature at 80 ��C for 10 min. Following overnight hybridization at 37 ��C, slides were washed three times with 50% formamide/2�� standard saline citrate at 45 ��C for 5 min each, followed by three washes with 1�� standard saline citrate (prewarmed to 60 ��C) at 45 ��C for 5 min each. Detection was then performed using a TSA kit (Molecular Probes, cat. no. “type”:”entrez-nucleotide”,”attrs”:”text”:”T20931″,”term_id”:”2756849″,”term_text”:”T20931″T20931) was then performed.

In a retrospective

In a retrospective Idelalisib CLL review, systemic chemotherapy demonstrated the ability to convert 13% of patients with initially unresectable CRLM to resectable status with a postoperative 5-year survival rate of 33%.9 Other reports have also demonstrated the use of chemotherapy regimens to downstage the size of tumours in order to convert unresectable tumours to resectable status. The recently published Eloxatin? for peri-operative use (EPOC) trial (European Organization for Research and Treatment of Cancer [EORTC] 40893), conducted by Nordlinger’s group, is the first randomized trial of perioperative chemotherapy to demonstrate a progression-free survival advantage for patients using this strategy.10 However, other publications highlight the liver toxicities and potential perioperative morbidity and mortality associated with preoperative chemotherapy.

11,12 Another potential advantage of perioperative chemotherapy was recently proposed by Blazer et al. from MD Anderson Cancer Center.13 In their paper, the authors highlight the importance of response to preoperative chemotherapy as a predictor of postoperative outcome. It would therefore seem logical to choose the chemotherapy with the highest response rate in the preoperative setting. The perioperative use of bevacizumab ([Bev] Avastin?; Hoffmann-Laroche, Basel, Switzerland), a humanized monoclonal antibody that targets vascular endothelial growth factor (VEGF), remains controversial. Although the survival benefit of Bev used in the palliative setting has been established,14 its impact on response rate is not as clear as recent data from the NO16966 trial failed to show an increased response rate when Bev was combined with FOLFOX-4.

15 Despite this, Bev is commonly used in combination with cytotoxic chemotherapy before surgical resection in patients with CRLM. Because of reports of haematologic Anacetrapib toxicity, intestinal perforation and an increased risk of postoperative bleeding, surgeons and oncologists have been reluctant to use Bev in the perioperative setting. However, a retrospective study from the MD Anderson showed no significant increase in hepatobiliary or wound complications, and no postoperative deaths in patients who received preoperative Bev.16 In the same series, only 12% of patients experienced preoperative adverse events related to Bev therapy. Other series have also confirmed that the neoadjuvant use of Bev in combination with cytotoxic agents did not significantly increase postoperative complications.17�C19 In the current study, we review our experience with a Bev and oxaliplatin-based chemotherapy regimen administered to patients with CRLM perioperatively in order to determine the safety and efficacy of this regimen and its impact on survival.

The trends in market share for adults for a wider range of brands

The trends in market share for adults for a wider range of brands (which are not presented in a table here) are similar to those for adolescents shown in www.selleckchem.com/products/CP-690550.html Table 3. However, older adult smokers were less likely than younger ones to change their brand preference. Discussion The menthol cigarette market in Australia was highly gendered prior to its decline, with menthol brands being much more popular among female than male smokers in all age groups in the early 1980s. The fundamental shift in the Australian market we found can be summarized as markedly declining numbers of younger smokers experimenting with and developing settled preferences for menthol brands, while they retained their popularity among older women.

We found that the market share of Alpine among Australian adolescents declined substantially between 1984 and 2008, with most of the decline occurring by 1996. Market share of Alpine also declined among adult smokers between 1980 and 2009. The decline in use of Alpine among adults occurred within the context of a general decline in menthol smoking in Australia rather than Alpine primarily losing market share to other menthol brands or brand varieties (as is shown the lack of any temporal trend in the proportion of menthol market share held by Alpine). Due to limitations of the available data, we are unable to exclude the possibility that Alpine lost market share to menthol varieties of major brand families among adolescents rather than to nonmenthol brands. We also do not know whether the market shares of either Alpine or all menthol brands peaked prior to the study period.

However, our results suggest a process of declining popularity beginning among adult smokers and then following among adolescents in the mid-1980s. Brefeldin_A Our results suggest that menthol cigarettes once functioned as starter cigarettes in Australia for a proportion of adolescents but are no longer an important starter product for either sex. In contrast with adolescents in the United States, where nearly half of adolescents smoke menthols (Hersey et al., 2006; Hersey, Nonnemaker and Homsi (2010); Kreslake et al., 2008), only a small proportion of Australian adolescents smoked menthol cigarettes at any point in the past two decades. Further, the decline in the popularity of Alpine among adolescents preceded the recent decline in smoking prevalence among adolescents, which began at least one survey wave (3 years) later. While menthol cigarettes may have an important function as starter cigarettes within particular contexts, such as in the United States, they are no longer an important part of the uptake process in Australia.

24 [1 18, 1 30], p < 001; lifetime: OR = 1 25

24 [1.18, 1.30], p < .001; lifetime: OR = 1.25 blog of sinaling pathways [1.19, 1.30], p < .001). For males, increasing SS by one point and one SD increased the odds of past 30-day smoking by 39% (lifetime, 36%) and 253% (lifetime 250%), respectively; a male participant with the maximum SS score of 16 was about 192 (lifetime 131) times more likely to have smoked in the past 30 days than one with the minimum score of 0. For females, one point and one SD increases in SS were associated with 24% and 138% increases in the probability of past 30-day smoking and 25% and 147% in the probability of lifetime smoking. A female participant with the maximum SS score was 29 times more likely to have smoked in the past 30 days and 34 times more likely in her lifetime, than one with the minimum score.

Simple Mediational Models Results from the simple meditational models are shown in Table 2. For both 30-day smoking models, the a (associations between SS and mediators) and b (associations between mediators and smoking) paths were significant. We found that both negative affect (ab = 0.11, 95% CI 0.09, 0.14) and risk perceptions (ab = 0.06, 95% CI 0.03, 0.09) were significant mediators of the relationship between SS and past 30-day smoking. For lifetime smoking, the a and b paths were again significant for both models. Additionally, negative affect (ab = 0.05, 95% CI 0.03, 0.06) and risk perceptions (ab = 0.06, 95% CI 0.04, 0.08) each mediated the relationship between SS and lifetime smoking. Table 2.

Mediational Models for Past 30-Day and Lifetime Smoking Multiple Mediation Model To assess the effects of negative affect and risk perceptions together, we then fit multiple mediation models for both 30-day and lifetime smoking (see Table 3 and Figure 1). For past 30-day smoking, the specific indirect effects of both negative affect (z = 3.40, p < .001) and risk perceptions (z = 4.33, p < .001) were significant. The combined indirect effect was also significant (z = 5.38, p < .001). Similarly, for the lifetime smoking model, indirect effects of negative affect (z = 3.25, p = .001) and risk perceptions (z = 4.51, p < .001) and the combined indirect effect (z = 6.14, p < .001) were all significant. The indirect effects of negative affect and risk perceptions were not significantly Dacomitinib different from each other in either model. Table 3. Multiple Mediation Model of the Indirect Relationship Between Sensation Seeking and Past 30-Day and Lifetime Smoking Figure 1. Indirect effects of sensation seeking on past 30-day and lifetime smoking through negative affect and risk perceptions. Note: Regression coefficients (standard errors) depicted above each arrow are for past 30-day smoking; coefficients (standard errors) …

The IC50 of recombinant HM103p18 as assayed by Cyclin D/CDK4-depe

The IC50 of recombinant HM103p18 as assayed by Cyclin D/CDK4-dependent full read Rb phosphorylation was higher than others have reported for purified p18INK4c protein.33,34 Therefore, some improvements in formulation and/or modifications of HM103p18 by using different MTDs and proteins with and without His tag or NLS sequences might further enhance therapeutic activity of the protein. Finally, we expect improvements to protein refolding and solution-formulation will enhance stability, reduce aggregation, and/or increase fill-finish concentration. In conclusion, this pilot study suggests that HM103p18 could have antitumor therapeutic activity as well as additional therapeutic applications by modulating the proliferation, differentiation and survival of stem cells with specific requirements for CDK4/6.

Materials and Methods Preparation of recombinant p18INK4c fusion proteins. MTD sequences, including MTD103, were identified from a screen of 1,500 signal peptides for sequences with protein transduction activity and were subsequently modified (D. Jo et al., manuscript in preparation). Coding sequences for p18INK4c and EGFP fusion proteins were cloned into pET-28a(+) (Novagen, Darmstadt, Germany) from PCR-amplified DNA segments (Supplementary Table S1). EGFP fusion proteins included a positive control containing the MTS from FGF4 (AAVLLPVLLAAP) and an arbitrary peptide (SANVEPLERL) that served as a negative control. Hp18 consists of an amino terminal 6x histidine tag (MGSSHHHHHHSSLVPRGSH) and NLS (KKKRK) from SV40 large T antigen appended to the human p18INK4c sequence (residues 2�C168).

HMTD103p18, HTatp18, Hphp18, and HAntp18, are identical to Hp18 but contain the hydrophobic MTD103 sequence or protein transduction domains from HIV-I Tat (YGRKKRRQRRR), human Hph-1 (YARVRRRGPRR) and Drosophila Ant (RQIKIWFQNRRMKWKK), respectively (Supplementary Tables S2 and S3). The recombinant proteins were purified from Escherichia coli BL21-CodonPlus (DE3) cells grown to an A600 of 0.6 and induced for 3 hours with 0.6 mmol/l IPTG. Denatured recombinant proteins were purified by Ni2+ affinity chromatography as directed by the supplier (Qiagen, Hilden, Germany). After purification, they were dialyzed against a refolding buffer (0.55 mol/l guanidine HCl, 0.44 mol/l L-arginine, 50 mmol/l Tris�CHCl, 150 mmol/l NaCl, 1 mmol/l EDTA, 100 mmol/l NDSB, 2 mmol/l reduced glutathione, and 0.

2 mmol/l oxidized glutathione) and changed to a physiological buffer such as RPMI 1640 medium. The CKI activity of purified Hp18 and HM103p18 proteins was determined by measuring the inhibition of Rb phosphorylation by CDK6/Cyclin D1 kinase, according to the manufacturer’s instructions (Cell Signaling, Danvers, MA). The structures of MTD- and Entinostat PTD-containing p18INK4c proteins were modeled using the AMBER 10.035,36 package on a KIST supercomputer based on a continuum solvation model.