Phosphorylation by Cdk2 is required for Myc to repress Ras-induced senescence in cotransformation.

The MYC and RAS oncogenes are frequently activated in cancer and, together, are sufficient to transform rodent cells. The basis for this cooperativity remains unclear. We found that although Ras interfered with Myc-induced apoptosis, Myc repressed Ras-induced senescence, together abrogating two main barriers of tumorigenesis. Inhibition of cellular senescence required phosphorylation of Myc at Ser-62 by cyclin E/cyclin-dependent kinase (Cdk) 2. Cdk2 interacted with Myc at promoters, where it affected Myc-dependent regulation of genes, including Bmi-1, p16, p21, and hTERT, which encode proteins known to control senescence. Repression of senescence by Myc was abrogated by the Cdk inhibitor p27Kip1, which is induced by antiproliferative signals like IFN-gamma or by pharmacological inhibitors of Cdk2 but not by inhibitors of other Cdks. In contrast, a phospho-mimicking Myc-S62D mutant was resistant to these manipulations. Inhibition of cyclin E/Cdk2 reversed the senescence-associated gene expression pattern imposed by Myc/cyclin E/Cdk2. This indicates a role of Cdk2 as a transcriptional cofactor and activator of the antisenescence function of Myc and provides mechanistic insight into the Myc-p27Kip1 antagonism. Finally, our findings highlight that pharmacological inhibition of Cdk2 activity is a potential therapeutical principle for cancer therapy, in particular for tumors with activated Myc or Ras.

Regulation of the transcription factor FOXM1c by Cyclin E/CDK2.

The FOXM1 forkhead proteins, originally identified as M-phase phosphoproteins, are proliferation-associated transcriptional regulators involved in cell cycle progression, genetic stability and tumorigenesis. Here we demonstrate that Cyclin-dependent kinases regulate the transcriptional activity of FOXM1c. This is independent of an N-terminal negative regulatory domain and of the forkhead DNA binding domain. Instead we mapped the responsive sites in the transactivation domain. A combination of three phosphorylation sites mediates the Cyclin E and Cyclin A/CDK2 effects. Our findings provide evidence for a novel Cyclin E/CDK2 substrate that functions in cell cycle control.

The protein kinase DYRK1A phosphorylates the splicing factor SF3b1/SAP155 at Thr434, a novel in vivo phosphorylation site.

BACKGROUND: The U2 small nuclear ribonucleoprotein particle (snRNP) component SF3b1/SAP155 is the only spliceosomal protein known to be phosphorylated concomitant with splicing catalysis. DYRK1A is a nuclear protein kinase that has been localized to the splicing factor compartment. Here we describe the identification of DYRK1A as a protein kinase that phosphorylates SF3b1 in vitro and in cultivated cells. RESULTS: Overexpression of DYRK1A caused a markedly increased phosphorylation of SF3b1 in COS-7 cells as assessed by Western blotting with an antibody specific for phosphorylated Thr-Pro dipeptide motifs. Phosphopeptide mapping of metabolically labelled SF3b1 showed that the majority of the in vivo-phosphopeptides corresponded to sites also phosphorylated by DYRK1A in vitro. Phosphorylation with cyclin E/CDK2, a kinase previously reported to phosphorylate SF3b1, generated a completely different pattern of phosphopeptides. By mass spectrometry and mutational analysis of SF3b1, Thr434 was identified as the major phosphorylation site for DYRK1A. Overexpression of DYRK1A or the related kinase, DYRK1B, resulted in an enhanced phosphorylation of Thr434 in endogenous SF3b1 in COS-7 cells. Downregulation of DYRK1A in HEK293 cells or in HepG2 cells by RNA interference reduced the phosphorylation of Thr434 in SF3b1. CONCLUSION: The present data show that the splicing factor SF3b1 is a substrate of the protein kinase DYRK1A and suggest that DYRK1A may be involved in the regulation of pre mRNA-splicing.

ING5 is phosphorylated by CDK2 and controls cell proliferation independently of p53.

Inhibitor of growth (ING) proteins have multiple functions in the control of cell proliferation, mainly by regulating processes associated with chromatin regulation and gene expression. ING5 has been described to regulate aspects of gene transcription and replication. Moreover deregulation of ING5 is observed in different tumors, potentially functioning as a tumor suppressor. Gene transcription in late G1 and in S phase and replication is regulated by cyclin-dependent kinase 2 (CDK2) in complex with cyclin E or cyclin A. CDK2 complexes phosphorylate and regulate several substrate proteins relevant for overcoming the restriction point and promoting S phase. We have identified ING5 as a novel CDK2 substrate. ING5 is phosphorylated at a single site, threonine 152, by cyclin E/CDK2 and cyclin A/CDK2 in vitro. This site is also phosphorylated in cells in a cell cycle dependent manner, consistent with it being a CDK2 substrate. Furthermore overexpression of cyclin E/CDK2 stimulates while the CDK2 inhibitor p27KIP1 represses phosphorylation at threonine 152. This site is located in a bipartite nuclear localization sequence but its phosphorylation was not sufficient to deregulate the subcellular localization of ING5. Although ING5 interacts with the tumor suppressor p53, we could not establish p53-dependent regulation of cell proliferation by ING5 and by phospho-site mutants. Instead we observed that the knockdown of ING5 resulted in a strong reduction of proliferation in different tumor cell lines, irrespective of the p53 status. This inhibition of proliferation was at least in part due to the induction of apoptosis. In summary we identified a phosphorylation site at threonine 152 of ING5 that is cell cycle regulated and we observed that ING5 is necessary for tumor cell proliferation, without any apparent dependency on the tumor suppressor p53.

A preliminary report on stem cell therapy for neuropathic pain in humans.

OBJECTIVE: Mesenchymal stem cells (MSCs) have been shown in animal models to attenuate chronic neuropathic pain. This preliminary study investigated if: i) injections of autologous MSCs can reduce human neuropathic pain and ii) evaluate the safety of the procedure. METHODS: Ten subjects with symptoms of neuropathic trigeminal pain underwent liposuction. The lipoaspirate was digested with collagenase and washed with saline three times. Following centrifugation, the stromal vascular fraction was resuspended in saline, and then transferred to syringes for local injections into the pain fields. Outcome measures at 6 months assessed reduction in: i) pain intensity measured by standard numerical rating scale from 0-10 and ii) daily dosage requirements of antineuropathic pain medication. RESULTS: Subjects were all female (mean age 55.3 years ± standard deviation [SD] 14.67; range 27-80 years) with pain symptoms lasting from 4 months to 6 years and 5 months. Lipoaspirate collection ranged from 102-214 g with total cell numbers injected from 33 million to 162 million cells. Cell viability was 62%-91%. There were no systemic or local tissue side effects from the stem cell therapy (n=41 oral and facial injection sites). Clinical pain outcomes showed that at 6 months, 5/9 subjects had reduced both pain intensity scores and use of antineuropathic medication. The mean pain score pre-treatment was 7.5 (SD 1.58) and at 6 months had decreased to 4.3 (SD 3.28), P=0.018, Wilcoxon signed-rank test. Antineuropathic pain medication use showed 5/9 subjects reduced their need for medication (gabapentin, P=0.053, Student's t-test). CONCLUSION: This preliminary open-labeled study showed autologous administration of stem cells for neuropathic trigeminal pain significantly reduced pain intensity at 6 months and is a safe and well tolerated intervention.

Equivalence of quality control strains of microorganisms used in the compendial microbiological tests: are national culture collection strains identical?

The pharmacopoeias list a number of microorganisms to be used in the compendial microbiological tests for confirming the growth-promoting, indicative, and inhibitory properties of the media and demonstrating the suitability of the test for a specific test article. Major national culture collections are specified as the sources for these test strains based on their history of deposition and maintenance and use in the compendial tests. Using these microorganisms, it has long been assumed that these strains are interchangeable and that sourcing the strains from different culture collections has no impact on the result of the media quality control and method qualification tests. In order to evaluate whether this assumption is correct and to add more certainty to the procedures, we investigated whether there are detectable differences among isolates of the same strain sourced from different culture collections. Using various phenotypic and genotypic identification and strain typing methods, nine major pharmacopoeial species were analyzed. As expected, most of the species showed very uniform patterns across the isolates, indicating that the strains were indeed identical. Surprisingly, the strains of Salmonella enterica subsp. enterica serotype abony showed distinct differences at both the genotypic and the phenotypic level, suggesting that the strains sourced from the different culture collections were not identical strains, or that they have undergone detectable genetic shift from the time they were derived from the original depositor. Irrespective of the level of genotypic or phenotypic homology identified here, there are no practical consequences on their performance in compendial assays. It is concluded that the compendial strains investigated in this study are indeed equivalent and will perform identically in compendial tests, making it safe to base pharmaceutical quality control procedures on the strains sourced from any of the recognized national culture collections.

The regulation of SIRT2 function by cyclin-dependent kinases affects cell motility.

Cyclin-dependent kinases (Cdks) fulfill key functions in many cellular processes, including cell cycle progression and cytoskeletal dynamics. A limited number of Cdk substrates have been identified with few demonstrated to be regulated by Cdk-dependent phosphorylation. We identify on protein expression arrays novel cyclin E-Cdk2 substrates, including SIRT2, a member of the Sirtuin family of NAD(+)-dependent deacetylases that targets alpha-tubulin. We define Ser-331 as the site phosphorylated by cyclin E-Cdk2, cyclin A-Cdk2, and p35-Cdk5 both in vitro and in cells. Importantly, phosphorylation at Ser-331 inhibits the catalytic activity of SIRT2. Gain- and loss-of-function studies demonstrate that SIRT2 interfered with cell adhesion and cell migration. In postmitotic hippocampal neurons, neurite outgrowth and growth cone collapse are inhibited by SIRT2. The effects provoked by SIRT2, but not those of a nonphosphorylatable mutant, are antagonized by Cdk-dependent phosphorylation. Collectively, our findings identify a posttranslational mechanism that controls SIRT2 function, and they provide evidence for a novel regulatory circuitry involving Cdks, SIRT2, and microtubules.

The human trithorax protein hASH2 functions as an oncoprotein.

Regulation of chromatin is an important aspect of controlling promoter activity and gene expression. Posttranslational modifications of core histones allow proteins associated with gene transcription to access chromatin. Closely associated with promoters of actively transcribed genes, trimethylation of histone H3 at lysine 4 (H3K4me3) is a core histone mark set by several protein complexes. Some of these protein complexes contain the trithorax protein ASH2 combined with the MLL oncoproteins. We identified human ASH2 in a complex with the oncoprotein MYC. This finding, together with the observation that hASH2 interacts with MLL, led us to test whether hASH2 itself is involved in transformation. We observed that hASH2 cooperates with Ha-RAS to transform primary rat embryo fibroblasts (REF). Furthermore, transformation of REFs by MYC and Ha-RAS required the presence of rAsh2. In an animal model, the hASH2/Ha-RAS-transformed REFs formed rapidly growing tumors characteristic of fibrosarcomas that, compared with tumors derived from MYC/Ha-RAS transformed cells, were poorly differentiated. This finding suggests that ASH2 functions as an oncoprotein. Although hASH2 expression at the mRNA level was generally not deregulated, hASH2 protein expression was increased in most human tumors and tumor cell lines. In addition, knockdown of hASH2 inhibited tumor cell proliferation. Taken together, these observations define hASH2 as a novel oncoprotein.

Mad1 function in cell proliferation and transcriptional repression is antagonized by cyclin E/CDK2.

The transcription factors of the Myc/Max/Mad network play essential roles in the regulation of cellular behavior. Mad1 inhibits cell proliferation by recruiting an mSin3-corepressor complex that contains histone deacetylase activity. Here we demonstrate that Mad1 is a potent inhibitor of the G(1) to S phase transition, a function that requires Mad1 to heterodimerize with Max and to bind to the corepressor complex. Cyclin E/CDK2, but not cyclin D and cyclin A complexes, fully restored S phase progression. In addition inhibition of colony formation and gene repression by Mad1 were also efficiently antagonized by cyclin E/CDK2. This was the result of cyclin E/CDK2 interfering with the interaction of Mad1 with HDAC1 and reducing HDAC activity. Our findings define a novel interplay between the cell cycle regulator cyclin E/CDK2 and Mad1 and its associated repressor complex and suggests an additional mechanism how cyclin E/CDK2 affects the G(1) to S phase transition.

Stimulation of c-MYC transcriptional activity and acetylation by recruitment of the cofactor CBP.

The c-MYC oncoprotein regulates various aspects of cell behaviour by modulating gene expression. Here, we report the identification of the cAMP-response-element-binding protein (CBP) as a novel c-MYC binding partner. The two proteins interact both in vitro and in cells, and CBP binds to the carboxy-terminal region of c-MYC. Importantly, CBP, as well as p300, is associated with E-box-containing promoter regions of genes that are regulated by c-MYC. Furthermore, c-MYC and CBP/p300 function synergistically in the activation of reporter-gene constructs. Thus, CBP and p300 function as positive cofactors for c-MYC. In addition, c-MYC is acetylated in cells. This modification does not require MYC box II, suggesting that it is independent of TRRAP complexes. Instead, CBP acetylates c-MYC in vitro, and co-expression of CBP with c-MYC stimulates in vivo acetylation. Functionally, this results in a decrease in ubiquitination and stabilization of c-MYC proteins. Thus, CBP and p300 are novel functional binding partners of c-MYC.

Expression profiling of breast cancer cells by differential peptide display.

Expression profiling of RNAs or proteins has become a promising means to investigate the heterogeneity of histopathologically defined classes of cancer. Peptides, representing degradation as well as processing products of proteins offer an even closer insight into cell physiology. Peptides are related to the turnover of cellular proteins and are capable to reflect disease-related changes in homoeostasis of the human body. Furthermore, peptides derived from tumor cells are potentially useful markers in the early detection of cancer. In this study, we introduced a method called differential peptide display (DPD) for separating, detecting, and identifying native peptides derived from whole cell extracts. This method is a highly standardized procedure, combining the power of reversed-phase chromatography with mass spectrometry. This technology is suitable to analyze cell lines, various tissue types and human body fluids. Peptide-based profiling of normal human mammary epithelial cells (HMEC) and the breast cancer cell line MCF-7 revealed complex peptide patterns comprising of up to 2300 peptides. Most of these peptides were common to both cell lines whereas about 8% differed in their abundance. Several of the differentially expressed peptides were identified as fragments of known proteins such as intermediate filament proteins, thymosins or Cathepsin D. Comparing cell lines with native tumors, overlapping peptide patterns were found between HMEC and a phylloides tumor (CP) on the one hand and MCF-7 cells and tissue from a invasive ductal carcinoma (DC) on the other hand.