Frap, FKBP12 rapamycin-associated protein, is a candidate gene for the plasmacytoma resistance locus Pctr2 and can act as a tumor suppressor gene

Susceptibility to mouse plasmacytomagenesis is a complex genetic trait controlled by several Pctr loci (Pctr1, Pctr2, etc). Congenic strain analysis narrowed the genetic interval surrounding the Pctr2 locus, and genes identified in the interval were sequenced from susceptible BALB/c and resistant DBA/2 mice. Frap (FKBP12 rapamycin-associated protein, mTOR, RAFT) was the only gene differing in amino acid sequence between alleles that correlated with strain sensitivity to tumor development. The in vitro kinase activity of the BALB/c FRAP allele was lower than the DBA/2 allele; phosphorylation of p53 and PHAS1/4EBP1 (properties of heat and acid stability/eukaryotic initiation factor 4E-binding protein) and autophosphorylation of FRAP were less efficient with the BALB/c allele. FRAP also suppressed transformation of NIH 3T3 cells by ras, with DBA/2 FRAP being more efficient than BALB/c FRAP. Rapamycin, a specific inhibitor of FRAP, did not inhibit growth of plasmacytoma cell lines. These studies identify Frap as a candidate tumor suppressor gene, in contrast to many reports that have focused on its prooncogenic properties. Frap may be similar to Tgfb and E2f in exerting both positive and negative growth-regulatory signals, depending on the timing, pathway, or tumor system involved. The failure of rapamycin to inhibit plasma cell tumor growth suggests that FRAP antagonists may not be appropriate for the treatment of plasma cell tumors. Pctr2 joins Pctr1 in possessing alleles that modify susceptibility to plasmacytomagenesis by encoding differences in efficiency of function (efficiency alleles), rather than all-or-none, gain-of-function, or loss-of-function alleles. By analogy, human cancer may also result from the combined effects of several inefficient alleles.     

A quantitative model for age-dependent expression of the p16INK4a tumor suppressor

Recent work has shown that expression of the p16^INK4a tumor suppressor increases with chronological age. Expression is accelerated by gerontogenic behaviors such as tobacco use and physical inactivity, and is also influenced by allelic genotype of a polymorphic single nucleotide polymorphism (SNP) rs10757278 that is physically linked with the p16^INK4a ORF. To understand the relationship between p16^INK4a expression, chronologic age, subject characteristics and host genetics, we sought to develop a mathematical model that links p16^INK4a expression with aging. Using an annotated dataset of 170 healthy adults for whom p16^INK4a expression and subject genotypes were known, we developed two alternative stochastic models that relate p16^INK4a expression to age, smoking, exercise and rs10757278 genotype. Levels of p16^INK4a increased exponentially and then saturated at later chronologic ages. The model, which best fit the data, suggests saturation occurs because of p16^INK4a-dependent attrition of subjects at older chronologic ages, presumably due to death or chronic illness. An important feature of our model is that factors that contribute to death in a non p16^INK4a-dependent manner do not affect our analysis. Interestingly, tobacco-related increases in p16^INK4a expression are predicted to arise from a decrease in the rate of p16^INK4a-dependent death. This analysis is most consistent with the model that p16^INK4a expression monotonically increases with age, and higher expression is associated with increased subject attrition.     

Frequent deletion of p16INK4a/MTS1 and p15INK4b/MTS2 in pediatric acute lymphoblastic leukemia

The tandemly linked p16INK4aMTS1 and p15INK4b/MTS2 genes on chromosome 9, band p21 encode proteins that function as specific inhibitors of the cyclin D-dependent kinases CDK4 and CDK6. This locus undergoes frequent bi-allelic deletion in human cancer cell lines, suggesting that the encoded proteins may function as tumor suppressors. However, more recent analysis of primary tumor samples has shown a much lower frequency of abnormalities affecting this region, raising doubt over the importance of these proteins in human malignancies. Hemizygous deletions and rearrangements of chromosome 9, band p21, are among the most frequent cytogenetic abnormalities detected in pediatric acute lymphoblastic leukemia (ALL), occurring in approximately 10% of cases. To determine if the p16INK4a/p15INK4b locus might be the target of these chromosomal lesions, we analyzed both genes in primary clinical samples from 43 pediatric ALL patients using interphase fluorescence in situ hybridization, Southern blot analysis, and the polymerase chain reaction. Deletions of p16INK4a/p15INK4b were identified in 18 of 20 cases with cytogenetically observed abnormalities of 9p and 5 of 23 with apparently normal chromosomes 9p, with the majority containing bi- allelic deletions (16 homozygous/7 hemizygous). Although most homozygous deletions involved both genes, Southern blot analysis showed an interstitial deletion in a single case that was confined to p16INK4a, suggesting that p15INK4b was not the critical target gene in this case. Sequence analysis of both p16INK4a and p15INK4b in all seven cases with hemizygous deletions failed to show mutations within the coding regions of the retained alleles. In this select group of patients, deletion of p16INK4a/p15INK4b was associated with T-cell phenotype, nonhyperdiploid karyotype (< 50 chromosomes), and poor event- free survival. These findings indicate that deletion of the p16INK4a/p15INK4b locus is one of the most common genetic abnormalities so far detected in pediatric ALL, and that loss of one or more of these cell cycle kinase inhibitors is important in leukemogenesis.     

p16INK4/p15INK4B gene inactivation is a frequent event in malignant T-cell lines

Abstract: The cell cycle regulators p16^INK4 and p15^INK4B have been mapped to the minimal region of overlap for chromosome 9p21 deletions, observed in a number of malignancies, suggesting that they could be tumor suppressor genes (TSGs). In the case of pl6^INK4 this has been further substantiated by the finding of small intragenic mutations. In this study we have investigated the p16^INK4 and p15^INK4B genes in 16 malignant T-cell lines by means of Southern blot, PCR and sequence analysis. p16^INK4 allelic deletions occurred in 15 of 16 cell lines; 12 of which were homozygous and 3 hemizygous. In 1 cell line (DND 41) the remaining p16^INK4 allele carried a microdeletion of 29 bp of exon 2, supporting the concept that p16^INK4 is a target TSG for deletions on 9p21. Most p16^INK4 deletions also included the p15^INK4B gene. However, 4 of the cell lines deleted for p16^INK4 showed no evidence of p15^INK4B loss, indicating that p15^INK4B is not the target in these cell lines.     

Histone deacetylase inhibitor suppression of autoantibody‐mediated arthritis in mice via regulation of p16INK4a and p21WAF1/Cip1 expression

Objective

To examine whether depsipeptide (FK228), a histone deacetylase (HDA) inhibitor, has inhibitory effects on the proliferation of synovial fibroblasts from rheumatoid arthritis (RA) patients, and to examine the effects of systemic administration of FK228 in an animal model of arthritis.

Methods

Autoantibody‐mediated arthritis (AMA) was induced in 19 male DBA/1 mice (6–7 weeks old); 10 of them were treated by intravenous administration of FK228 (2.5 mg/kg), and 9 were used as controls. The effects of FK228 were examined by radiographic, histologic, and immunohistochemical analyses and arthritis scores. RA synovial fibroblasts (RASFs) were obtained at the time of joint replacement surgery. In vitro effects of FK228 on cell proliferation were assessed by MTT assay. Cell morphology was examined by light and transmission electron microscopy. The effects on the expression of the cell cycle regulators p16^INK4a and p21^WAF1/Cip1 were examined by real‐time polymerase chain reaction and Western blot analysis. The acetylation status of the promoter regions of p16^INK4a and p21^WAF1/Cip1 were determined by chromatin immunoprecipitation assay.

Results

A single intravenous injection of FK228 (2.5 mg/ml) successfully inhibited joint swelling, synovial inflammation, and subsequent bone and cartilage destruction in mice with AMA. FK228 treatment induced histone hyperacetylation in the synovial cells and decreased the levels of tumor necrosis factor α and interleukin‐1β in the synovial tissues of mice with AMA. FK228 inhibited the in vitro proliferation of RASFs in a dose‐dependent manner. Treatment of cells with FK228 induced the expression of p16^INK4a and up‐regulated the expression of p21^WAF1/Cip1. These effects of FK228 on p16^INK4a and p21^WAF1/Cip1 were related to the acetylation of the promoter region of the genes.

Conclusion

Our findings strongly suggest that systemic administration of HDA inhibitors may represent a novel therapeutic target in RA by means of cell cycle arrest in RASFs via induction of p16^INK4a expression and increase in p21^WAF1/Cip1 expression.

     

Silencing of the p18INK4c gene by promoter hypermethylation in Reed-Sternberg cells in Hodgkin lymphomas

p18INK4c is a cyclin-dependent kinase (CDK) inhibitor that interferes with the Rb-kinase activity of CDK6/CDK4. Disruption of p18INK4c in mice impairs B-cell terminal differentiation and confers increased susceptibility to tumor development; however, alterations of p18INK4c in human tumors have rarely been described. We used a tissue-microarray approach to analyze p18INK4c expression in 316 Hodgkin lymphomas (HLs). Nearly half of the HL cases showed absence of p18INK4c protein expression by Reed-Sternberg (RS) cells, in contrast with the regular expression of p18INK4c in normal germinal center cells. To investigate the cause of p18INK4c repression in RS cells, the methylation status of the p18INK4c promoter was analyzed by methylation-specific polymerase chain reaction (PCR) and bisulfite sequencing. Hypermethylation of the p18INK4c promoter was detected in 2 of 4 HL-derived cell lines, but in none of 7 non-Hodgkin lymphoma (NHL)-derived cell lines. We also detected p18INK4c hypermethylation, associated with absence of protein expression, in 5 of 26 HL tumors. The correlation of p18INK4c immunostaining with the follow-up of the patients showed shorter overall survival in negative cases, independent of the International Prognostic Score. These findings suggest that p18INK4c may function as a tumor suppressor gene in HL, and its inactivation may contribute to the cell cycle deregulation and defective terminal differentiation characteristic of the RS cells.     

Functional Genomics of the 9p21.3 Locus for Atherosclerosis: Clarity or Confusion?

The 9p21.3 locus was the first to yield to genome-wide association studies (GWAS) seeking common genetic variants predisposing to increased risk of coronary artery atherosclerotic disease (CAD). The 59 single nucleotide polymorphisms that show highest association with CAD are clustered in a region 100,000 to 150,000 base pairs 5′ to the cyclin-dependent kinase inhibitors CDKN2B (coding for p15^ink4b) and CDKN2A (coding for p16^ink4a and p14^ARF). This region also covers the 3′ end of a long noncoding RNA transcribed antisense to CDKN2B (CDKN2BAS, aka ANRIL for antisense noncoding RNA at the ink4 locus) whose expression has been linked to chromatin remodeling at the locus. Despite intensive investigation over the past 7 years, the functional significance of the 9p21.3 locus remains elusive. Other variants at this locus have been associated with glaucoma, glioma, and type 2 diabetes mellitus, diseases that implicate tissue-resident macrophages. Here, we review the evidence that genetic variants at 9p21.3 disrupt tissue-specific enhancers and propose new insights to guide future studies.     

Ly-4, A New Locus Determining a Lymphocyte Cell-Surface Alloantigen in Mice

A new locus is described that determines an alloantigen on the surface of lymphocytes. It differs from loci previously described in that the corresponding antibody, at least in most antisera, reacts almost exclusively with node lymphocytes, and weakly or not at all with thymuslymphocytes. Positive strains include C57BL/6, C57BL/10, C57L, C57BR/cd, and RF; negative strains include BALB/c, C3H, and SJL. The symbol Ly-4 is assigned, with the C57BL/6 allele being Ly-4^b, and the BALB/c allele Ly-4^a.     

Reduction in levels of the cyclin-dependent kinase inhibitor p27kip-1 coupled with transforming growth factor β neutralization induces cell-cycle entry and increases retroviral transduction of primitive human hematopoietic cells

Successful gene therapy depends on stable transduction of hematopoietic stem cells. Target cells must cycle to allow integration of Moloney-based retroviral vectors, yet hematopoietic stem cells are quiescent. Cells can be held in quiescence by intracellular cyclin-dependent kinase inhibitors. The cyclin-dependent kinase inhibitor p15^INK4B blocks association of cyclin-dependent kinase (CDK)4/cyclin D and p27^kip-1 blocks activity of CDK2/cyclin A and CDK2/cyclin E, complexes that are mandatory for cell-cycle progression. Antibody neutralization of β transforming growth factor (TGFβ) in serum-free medium decreased levels of p15^INK4B and increased colony formation and retroviral-mediated transduction of primary human CD34⁺ cells. Although TGFβ neutralization increased colony formation from more primitive, noncycling hematopoietic progenitors, no increase in M-phase-dependent, retroviral-mediated transduction was observed. Transduction of the primitive cells was augmented by culture in the presence of antisense oligonucleotides to p27^kip-1 coupled with TGFβ-neutralizing antibodies. The transduced cells engrafted immune-deficient mice with no alteration in human hematopoietic lineage development. We conclude that neutralization of TGFβ, plus reduction in levels of the cyclin-dependent kinase inhibitor p27, allows transduction of primitive and quiescent hematopoietic progenitor populations.     

p19Arf induces p53-dependent apoptosis during Abelson virus-mediated pre-B cell transformation

The Ink4a/Arf locus encodes p16^Ink4a and p19^Arf and is among the most frequently mutated tumor suppressor loci in human cancer. In mice, many of these effects appear to be mediated by interactions between p19^Arf and the p53 tumor-suppressor protein. Because Tp53 mutations are a common feature of the multistep pre-B cell transformation process mediated by Abelson murine leukemia virus (Ab-MLV), we examined the possibility that proteins encoded by the Ink4a/Arf locus also play a role in Abelson virus transformation. Analyses of primary transformants revealed that both p16^Ink4a and p19^Arf are expressed in many of the cells as they emerge from the apoptotic crisis that characterizes the transformation process. Analyses of primary transformants from Ink4a/Arf null mice revealed that these cells bypassed crisis. Because expression of p19^Arf but not p16 ^Ink4a induced apoptosis in Ab-MLV-transformed pre-B cells, p19^Arf appears to be responsible for these events. Consistent with the link between p19^Arf and p53, Ink4a/Arf expression correlates with or precedes the emergence of cells expressing mutant p53. These data demonstrate that p19^Arf is an important part of the cellular defense mounted against transforming signals from the Abl oncoprotein and provide direct evidence that the p19^Arf–p53 regulatory loop plays an important role in lymphoma induction.     

p12CDK2-AP1 inhibits breast cancer cell proliferation and in vivo tumor growth

Purpose

p12^CDK2-AP1 is a growth suppressor that negatively regulates cyclin-dependent kinase 2 (CDK2) activities and shows to interfere in DNA replication. Here, we aim to elucidate the role of p12^CDK2-AP1 in breast cancer progression.

Methods

Expression of p12^CDK2-AP1 protein was examined in 60 pairs of breast cancer specimens and adjacent non-tumor tissues using immunohistochemistry assay. Loss-of-function and gain-of-function analysis was performed on MCF-7 and MDA-MB-231 breast cancer cells. Routine assays including MTT, colony formation, flow cytometry, and tumorigenesis in nude mice were performed and cell cycle regulators were analyzed.

Results

p12^CDK2-AP1 was found to be significantly downregulated in 60 breast cancer tissues compared to corresponding non-tumorous tissues. The proliferation and colony formation ability was inhibited in cells that transduced with p12^CDK2-AP1 over-expression lentivirus, but enhanced in cells that transduced with p12^CDK2-AP1 RNAi lentivirus. p12^CDK2-AP1 over-expression led to G0/G1 phase arrest in the cell cycle and caused expression changes of cell cycle-related genes (CDK2, CDK4, p16^Ink4A, p21^Cip1/Waf1). Furthermore, p12^CDK2-AP1 over-expression inhibited in vivo tumor growth in immunodeficiency mice, supporting an inhibitory role for p12^CDK2-AP1 in breast cancer development.

Conclusions

As a cell cycle regulator, p12^CDK2-AP1 is involved in the development of breast cancer and maybe a potential therapeutic candidate to suppress tumorigenicity in breast cancer.     

Experimental murine infection with the diffuse cutaneous leishmaniasis strain, Isabel

Infection with the Isabel strain of Leishmania has been followed in several inbred strains of mice over an extended period. The mouse strains segregate into three major types with respect to susceptibility to infection: BALB/c, BALB.B and SWR/J are susceptible; DBA/1J is intermediate; and C57BL/6, C57BL/10, DBA/2J and B10.D2 are resistant. Infections with other leishmanial strains have been well-characterized in the BALB/c mice. Therefore, BALB/c mice were selected for extended studies. Progression of the disease was assessed by the following parameters: (1) numbers of parasites isolated from various tissues including footpad lesions, spleens, and lymph nodes and (2) the presence of metastatic lesions. This model should prove valuable in the study of the early immunological events in leishmaniasis.     

Expression of p16INK4a,p15INK4b,p21WAF1/Clip1 cell cycle inhibitors on blastic cells in patients with acute myeloblastic leukemia (AML) and acute lymphoblastic leukemia (ALL)

Cyclin-dependent kinases (cdk) play the important role in neoplastic transformation. Their activity depends on interaction with proteins called inhibitors. There are two groups of inhibitors: INK4 (p16INK4a, p15INK4b, p18INK4c, p19INK4d) and proteins p21WAF1/Clip1, p27Kip1, p57Kip2. Alteration of inhibitors expression was assessed in acute lymphoblastic leukemia (ALL) and in acute myeloblastic leukemia (AML), but the results are not clear. The aim of our study was to estimate p16INK4a, p15INK-4b, p21WAF1/Clip1 expression in blast cells in patients with AML and ALL by cytochemistry method and to compare with the result of treatment. Forty-two patients were included in the study, 23 with AML and 19 with ALL. Expression of inhibitors was considered as positive when detected in > 5% of blast cells. Complete remission (CR) rate in patients with positive expression p16INK4a and p15INK4b was statistically significantly higher than in patients with negative expression: for p16INK4a chi 2 = 7.78, p < 0.01, for p15INK4b, chi 2 with Yates' modification = 3.94, p < 0.05. There was no difference in CR rate in patients with positive and negative p21WAF1/Clip1 expression. Moreover the patients with simultaneous expression of three inhibitors reached CR more often than the others: chi 2 = 7.43, p = 0.01 for AML and chi 2 = 6.74, p < 0.01 for ALL. Our study indicates that estimation of p16INK4a, p15INK4b, p21WAF1/Clip1 expression in blast cells can be used as prognostic factor in acute leukemia.     

Angiotensin converting-enzyme inhibitor treatment reduces glomerular p16INK4 and p27Kip1 expression in diabetic BBdp rats

Aims/hypothesis. Renal hypertrophy occurs early in diabetes mellitus and precedes the development of glomerulosclerosis and tubulointerstitial fibrosis. We have previously shown that cultured mesangial cells exposed to high glucose are arrested in the G₁-phase of the cell cycle and undergo cellular hypertrophy. High glucose-mediated induction of p27^Kip1, an inhibitor of cyclin-dependent kinases, is essential in this process. Further investigations have also shown that p27^Kip1 and p21^Cip1, other cyclin-dependent kinase inhibitors, are up regulated in the kidneys of mice with Type I (insulin-dependent) as well as Type II (non-insulin-dependent) diabetes mellitus. Our study was undertaken to test a potential effect of short-term treatment with the angiotensin-converting enzyme inhibitor enalapril on the glomerular expression of the cyclin-dependent kinase inhibitors p16^INK4, p21^Cip1, and p27^Kip1 in BBdp rats, an autoimmune model of Type I diabetes.¶Methods. We evaluated p16^INK4, p21^Cip1, and p27^Kip1 protein expression in isolated glomeruli by western blots. We also assessed p27^Kip1 positive glomerular cells by immunohistochemistry.¶Results. Glomerular expression of all three cyclin-dependent kinase inhibitors were stimulated in BBdp rats compared with non-diabetic BBdr animals. Enalapril treatment for 3 weeks, started after the onset of diabetes, reduced the glomerular expression of p16^INK4 and p27^Kip1 but not of p21^Cip1. Enalapril also prevented the increase in kidney weights observed in BBdp rats but had no effect on systolic blood pressure or glucose concentrations.¶Conclusion/interpretation. Our data show that enalapril attenuates the glomerular expression of cyclin-dependent kinase inhibitors in diabetes and suggest a molecular mechanism of how angiotensin-converting enzyme inhibitors prevent renal hypertrophy in diabetes. [Diabetologia (1999) 42: 1425–1432]     

The influence of INK4 proteins on growth and self-renewal kinetics of hematopoietic progenitor cells

This study investigated the influence of expression of proteins of the INK4 family, particularly p16, on the growth and self-renewal kinetics of hematopoietic cells. First, retrovirus-mediated gene transfer (RMGT) was used to restore p16(INK4a) expression in the p16(INK4a)-deficient lymphoid and myeloid cell lines BV173 and K562, and it was confirmed that this inhibited their growth. Second, to sequester p16(INK4a) and related INK4 proteins, cyclin-dependent kinase 4 (CDK4) was retrovirally transduced into normal human CD34(+) bone marrow cells and then cultured in myeloid colony-forming cell (CFC) assays. The growth of CDK4-transduced colonies was more rapid; the cell-doubling time was reduced; and, upon replating, the colonies produced greater yields of secondary colonies than mock-untransduced controls. Third, colony formation was compared by marrow cells from p16(INK4a-/-) mice and wild-type mice. The results from p16(INK4a-/-) marrow were similar to those from CDK4-transduced human CFCs, in terms of growth rate and replating ability, and were partially reversed by RMGT of p16(INK4a). Lines of immature granulocytic cells were raised from 15 individual colonies grown from the marrow of p16(INK4a-/-) mice. These had a high colony-forming ability (15%) and replating efficiency (96.7%). The p16(INK4a-/-) cell lines readily became growth factor-independent upon cytokine deprivation. Taken together, these results demonstrate that loss of INK4 proteins, in particular p16(INK4a), increases the growth rate of myeloid colonies in vitro and, more importantly, confers an increased ability for clonal expansion on hematopoietic progenitor cells.     

Toll样受体在约氏疟原虫感染早期树突状细胞应答中的作用地位

目的探讨致死型约氏疟原虫(Plasmodium yoelii17XL,P.y17XL)感染早期,Toll样受体(Toll like receptor,TLR)在树突状细胞(dendritic cells,DCs)活化中的作用地位。方法用P.y17XL感染易感的BALB/c和抵抗的DBA/2小鼠,计数红细胞感染率;制备感染前和感染后第3d、5d小鼠脾细胞悬液,采用流式细胞分析技术检测两种小鼠感染不同时间脾细胞悬液中细胞内表达TLR9(Toll like receptor 9,TLR9)的DCs和细胞表面表达TLR4(Toll like receptor 4,TLR4)的DCs的百分含量。结果两种小鼠脾DCs细胞内TLR9的表达水平均于感染后第3d开始明显升高(P<0.01),在第5d达到最高水平(P<0.01),但两种小鼠相比无统计学意义。同时,两种小鼠DCs表面TLR4的表达水平均未见明显变化。结论在P.y17XL感染早期,TLR9可能是介导DCs活化的模式识别受体。