Karl Riabowol

DNA damage-inducible gene p33ING2 negatively regulates cell proliferation through acetylation of p53

The p33ING1 protein is a regulator of cell cycle, senescence, and apoptosis. Three alternatively spliced transcripts of p33ING1 encode p47ING1a, p33ING1b, and p24ING1c. We cloned an additional ING family member, p33ING2/ING1L. Unlike p33ING1b, p33ING2 is induced by the DNA-damaging agents etoposide and neocarzinostatin. p33ING1b and p33ING2 negatively regulate cell growth and survival in a p53-dependent manner through induction of G1-phase cell-cycle arrest and apoptosis. p33ING2 strongly enhances the transcriptional-transactivation activity of p53. Furthermore, p33ING2 expression increases the acetylation of p53 at Lys-382. Taken together, p33ING2 is a DNA damage-inducible gene that negatively regulates cell proliferation through activation of p53 by enhancing its acetylation.

Paternal age is positively linked to telomere length of children

Telomere length is linked to age‐associated diseases, with shorter telomeres in blood associated with an increased probability of mortality from infection or heart disease. Little is known about how human telomere length is regulated despite convincing data from twins that telomere length is largely heritable, uniform in various tissues during development until birth and variable between individuals. As sperm cells show increasing telomere length with age, we investigated whether age of fathers at conception correlated with telomere length of their offspring. Telomere length in blood from 125 random subjects was shown to be positively associated with paternal age (+22 bp yr−1, 95% confidence interval 5.2–38.3, P = 0.010), and paternal age was calculated to affect telomere length by up to 20% of average telomere length per generation. Males lose telomeric sequence faster than females (31 bp yr−1, 17.6–43.8, P < 0.0001 vs. 14 bp yr−1, 3.5–24.8, P < 0.01) and the rate of telomere loss slows throughout the human lifespan. These data indicate that paternal age plays a role in the vertical transmission of telomere length and may contribute significantly to the variability of telomere length seen in the human population, particularly if effects are cumulative through generations.

Gender and telomere length: Systematic review and meta-analysis

BACKGROUND It is widely believed that females have longer telomeres than males, although results from studies have been contradictory. METHODS We carried out a systematic review and meta-analyses to test the hypothesis that in humans, females have longer telomeres than males and that this association becomes stronger with increasing age. Searches were conducted in EMBASE and MEDLINE (by November 2009) and additional datasets were obtained from study investigators. Eligible observational studies measured telomeres for both females and males of any age, had a minimum sample size of 100 and included participants not part of a diseased group. We calculated summary estimates using random-effects meta-analyses. Heterogeneity between studies was investigated using sub-group analysis and meta-regression. RESULTS Meta-analyses from 36 cohorts (36,230 participants) showed that on average females had longer telomeres than males (standardised difference in telomere length between females and males 0.090, 95% CI 0.015, 0.166; age-adjusted). There was little evidence that these associations varied by age group (p=1.00) or cell type (p=0.29). However, the size of this difference did vary by measurement methods, with only Southern blot but neither real-time PCR nor Flow-FISH showing a significant difference. This difference was not associated with random measurement error. CONCLUSIONS Telomere length is longer in females than males, although this difference was not universally found in studies that did not use Southern blot methods. Further research on explanations for the methodological differences is required.

Three Yeast Proteins Related to the Human Candidate Tumor Suppressor p33ING1 Are Associated with Histone Acetyltransferase Activities

ABSTRACT Three Saccharomyces cerevisiae proteins (Yng1/YOR064c, Yng2/YHR090c, and Pho23) and two Schizosaccharomyces pombeproteins (Png1/CAA15917 and Png2/CAA21250) share significant sequence identity with the human candidate tumor suppressor p33ING1in their C-terminal regions. The homologous regions contain PHD finger domains which have been implicated in chromatin-mediated transcriptional regulation. We show that GFP-Yng2, like human Ing1, is localized in the nucleus. Deletion of YNG2 results in several phenotypes, including an abnormal multibudded morphology, an inability to utilize nonfermentable carbon sources, heat shock sensitivity, slow growth, temperature sensitivity, and sensitivity to caffeine. These phenotypes are suppressed by expression of either human Ing1 or S. pombe Png1, suggesting that the yeast and human proteins are functionally conserved. Yng1- and Pho23-deficient cells also share some of these phenotypes. We demonstrated by yeast two-hybrid and coimmunoprecipitation tests that Yng2 interacts with Tra1, a component of histone acetyltransferase (HAT) complexes. We further demonstrated by coimmunoprecipitation that HA-Yng1, HA-Yng2, HA-Pho23, and HA-Ing1 are associated with HAT activities in yeast. Genetic and biochemical evidence indicate that the Yng2-associated HAT is Esa1, suggesting that Yng2 is a component of the NuA4 HAT complex. These studies suggest that the yeast Ing1-related proteins are involved in chromatin remodeling. They further suggest that these functions may be conserved in mammals and provide a possible mechanism for the human Ing1 candidate tumor suppressor.

REAP: A two minute cell fractionation method

BackgroundThe translocation or shuttling of proteins between the nucleus and cytoplasm (nucleocytoplasmic transport [NCPT]) is often a rapid event following stimulation with growth factors or in response to stress or other experimental manipulations. Commonly used methods to separate nuclei from cytoplasm employ lengthy steps such as density gradient centrifugation which exposes cells to non-physiological hyperosmotic conditions for extended time periods resulting in varying degrees of leakage between the nucleus and cytoplasm. To help maintain and quantify nuclear:cytoplasmic ratios of proteins, agents such as leptomycin B have been employed to be able to better analyze NCPT by inhibiting nuclear export. To track NCPT in the absence of these experimental manipulations that could introduce unknown artefacts, we have developed a rapid method that appears to produce pure nuclear and cytoplasmic fractions, suitable for obtaining accurate estimates of the nuclear:cytoplasmic ratios of proteins known to undergo NCPT.FindingsWe have developed a R apid, E fficient A nd P ractical (REAP) method for subcellular fractionation of primary and transformed human cells in culture. The REAP method is a two minute non-ionic detergent-based purification technique requiring only a table top centrifuge, micro-pipette and micro-centrifuge tubes. This inexpensive method has proven to efficiently separate nuclear from cytoplasmic proteins as estimated by no detectible cross-contamination of the nucleoporin and lamin A nuclear markers or the pyruvate kinase and tubulin cytoplasmic markers. REAP fractions also mirrored TNFα induced NF-κB NCPT observed in parallel by indirect immunofluorescence.ConclusionsThis method drastically reduces the time needed for subcellular fractionation, eliminates detectable protein degradation and maintains protein interactions. The simplicity, brevity and efficiency of this procedure allows for tracking ephemeral changes in subcellular relocalization of proteins while maintaining protein integrity and protein complex interactions.

Different HATS of the ING1 gene family

The ING family of proteins are involved in chromatin remodelling, and bind to and affect the activity of histone acetyltransferase, histone deacetylase, and factor acetyltransferase protein complexes. Some family members affect transcription, including the expression of p53-inducible genes such as p21 and Bax, and ING2 induces p53 acetylation on a site implicated in the regulation of p53 activity. ING1 promotes DNA repair and interacts with proliferating cell nuclear antigen, thus linking DNA repair, apoptosis and chromatin remodelling. Here, we summarize what is known about the molecular interactions of ING1 family proteins and, based on these interactions, develop a model to better understand the impact of ING proteins on multiple biological processes.

Extension of the replicative life span of human diploid fibroblasts by inhibition of the p33ING1 candidate tumor suppressor

Previous studies suggest that tumor suppressors may play significant roles in blocking the growth of cells during cellular senescence. We therefore studied the potential involvement of a novel growth inhibitor and candidate tumor suppressor gene called ING1, which we have cloned recently (I. Garkavtsev, A. Kazarov, A. Gudkov, and K. Riabowol, Nat. Genet. 14:415-420, 1996), in the process of cellular senescence. Our results show that the RNA and protein levels of ING1 were 8- to 10-fold higher in senescent cells than in young, proliferation-competent human diploid fibroblasts. Expression of the nuclear p33ING1 protein was regulated during the cell cycle, reaching maximal levels during DNA synthesis. Chronic expression of antisense ING1 RNA reproducibly resulted in extension of the proliferative life span of normal human fibroblasts by approximately seven population doublings.

Suppression of ING1 expression in sporadic breast cancer

Down regulation of the ING1 candidate tumour suppressor promotes growth in soft agar and focus formation in vitro and tumour formation in vivo. ING1 encodes a nuclear, cell cycle-regulated protein, overexpression of which efficiently blocks cell growth and is capable of inducing apoptosis in different experimental systems. Here we present the first report of ING1 mutation and expression analysis in a total of 452 cancer samples. One germline missense alteration and three germline silent alterations were detected in 377 primary breast cancers while marked (2 – 10-fold) decreases in ING1 mRNA expression were seen in 44% of primary breast cancers and in ten of ten breast cancer cell lines examined. Furthermore, the majority of breast cancers (58%) showing decreased ING1 expression had metastasized to regional lymph nodes whereas only 9% of cancers with elevated ING1 expression, compared to adjacent normal tissues, were metastatic. Thus, ING1 mutation is very rare in breast or ovarian cancers, however, repression of ING1 expression frequently accompanies tumour development of breast cancer.

Human ING1 Proteins Differentially Regulate Histone Acetylation

ING1 proteins are nuclear, growth inhibitory, and regulate apoptosis in different experimental systems. Here we show that similar to their yeast homologs, human ING1 proteins interact with proteins associated with histone acetyltransferase (HAT) activity, such as TRRAP, PCAF, CBP, and p300. Human ING1 immunocomplexes contain HAT activity, and overexpression of p33ING1b, but not of p47ING1a, induces hyperacetylation of histones H3 and H4, in vitro and in vivo at the single cell level. p47ING1a inhibits histone acetylation in vitro and in vivo and binds the histone deacetylase HDAC1. Finally, we present evidence indicating that p33ING1b affects the degree of physical association between proliferating cell nuclear antigen (PCNA) and p300, an association that has been proposed to link DNA repair to chromatin remodeling. Together with the finding that human ING1 proteins bind PCNA in a DNA damage-dependent manner, these data suggest that ING1 proteins provide a direct linkage between DNA repair, apoptosis, and chromatin remodeling via multiple HAT·ING1·PCNA protein complexes.

Increased Expression of Cyclin D2 during Multiple States of Growth Arrest in Primary and Established Cells

ABSTRACT Cyclin D2 is a member of the family of D-type cyclins that is implicated in cell cycle regulation, differentiation, and oncogenic transformation. To better understand the role of this cyclin in the control of cell proliferation, cyclin D2 expression was monitored under various growth conditions in primary human and established murine fibroblasts. In different states of cellular growth arrest initiated by contact inhibition, serum starvation, or cellular senescence, marked increases (5- to 20-fold) were seen in the expression levels of cyclin D2 mRNA and protein. Indirect immunofluorescence studies showed that cyclin D2 protein localized to the nucleus in G0, suggesting a nuclear function for cyclin D2 in quiescent cells. Cyclin D2 was also found to be associated with the cyclin-dependent kinases CDK2 and CDK4 but not CDK6 during growth arrest. Cyclin D2-CDK2 complexes increased in amounts but were inactive as histone H1 kinases in quiescent cells. Transient transfection and needle microinjection of cyclin D2 expression constructs demonstrated that overexpression of cyclin D2 protein efficiently inhibited cell cycle progression and DNA synthesis. These data suggest that in addition to a role in promoting cell cycle progression through phosphorylation of retinoblastoma family proteins in some cell systems, cyclin D2 may contribute to the induction and/or maintenance of a nonproliferative state, possibly through sequestration of the CDK2 catalytic subunit.