A CANCER PERSISTENT DNA REPAIR CIRCUIT DRIVEN BY MDM2, MDM4 (MDMX), AND MUTANT P53 FOR RECRUITMENT OF MDC1 AND 53BP1 TO CHROMATIN.

The influence of the metastasis promoting proteins mutant p53 (mtp53) and MDM2 on (CPR) to promote cancer cell survival is understudied. Interactions between the DNA repair choice protein 53BP1 and wild type tumor suppressor protein p53 (wtp53) regulates cell cycle control. Cancer cells often express elevated levels of transcriptionally inactive missense mutant p53 (mtp53) that interacts with MDM2 and MDM4/MDMX (herein called MDMX).

Gain-of-Function Mutant p53 R273H Interacts with Replicating DNA and PARP1 in Breast Cancer.

Over 80% of triple-negative breast cancers (TNBC) express mutant p53 (mtp53) and some contain oncogenic gain-of-function (GOF) p53. We previously reported that GOF mtp53 R273H upregulates the chromatin association of mini chromosome maintenance (MCM) proteins MCM2-7 and PARP and named this the mtp53-PARP-MCM axis. In this study, we dissected the function and association between mtp53 and PARP using a number of different cell lines, patient-derived xenografts (PDX), tissue microarrays (TMA), and The Cancer Genome Atlas (TCGA) database.

Identification, validation, and targeting of the mutant p53-PARP-MCM chromatin axis in triple negative breast cancer.

Over 80% of triple negative breast cancers express mutant p53. Mutant p53 often gains oncogenic function suggesting that triple negative breast cancers may be driven by p53 protein type. To determine the chromatin targets of this gain-of-function mutant p53 we used inducible knockdown of endogenous gain-of-function mtp53 in MDA-MB-468 cells in conjunction with stable isotope labeling with amino acids in cell culture and subcellular fractionation.

Hot Spot Mutation in TP53 (R248Q) Causes Oncogenic Gain-of-Function Phenotypes in a Breast Cancer Cell Line Derived from an African American patient.

African American (AA) breast cancer patients often have triple negative breast cancer (TNBC) that contains mutations in the TP53 gene. The point mutations at amino acid residues R273 and R248 both result in oncogenic gain-of-function (GOF) phenotypes. Expression of mutant p53 (mtp53) R273H associates with increased cell elasticity, survival under serum deprivation conditions, and increased Poly (ADP ribose) polymerase 1 (PARP1) on the chromatin in the AA-derived TNBC breast cancer cell line MDA-MB-468.

Homozygous mdm2 SNP309 cancer cells with compromised transcriptional elongation at p53 target genes are sensitive to induction of p53-independent cell death.

A single nucleotide polymorphism (T to G) in the mdm2 P2 promoter, mdm2 SNP309, leads to MDM2 overexpression promoting chemotherapy resistant cancers. Two mdm2 G/G SNP309 cancer cell lines, MANCA and A875, have compromised wild-type p53 that co-localizes with MDM2 on chromatin. We hypothesized that MDM2 in these cells inhibited transcription initiation at the p53 target genes p21 and puma.

Proteome-wide analysis of mutant p53 targets in breast cancer identifies new levels of gain-of-function that influence PARP, PCNA, and MCM4.

The gain-of-function mutant p53 (mtp53) transcriptome has been studied, but, to date, no detailed analysis of the mtp53-associated proteome has been described. We coupled cell fractionation with stable isotope labeling with amino acids in cell culture (SILAC) and inducible knockdown of endogenous mtp53 to determine the mtp53-driven proteome. Our fractionation data highlight the underappreciated biology that missense mtp53 proteins R273H, R280K, and L194F are tightly associated with chromatin.

Impedimetric detection of mutant p53 biomarker-driven metastatic breast cancers under hyposmotic pressure.

In cancer cells, the oncogenic mutant p53 (mtp53) protein is present at high levels and gain-of-function (GOF) activities with more expression of mtp53 proteins contribute to tumor growth and metastasis. Robust analytical approaches that probe the degree of metastasis of cancer cells in connection with the mtp53 activity will be extremely useful not only for establishing a better cancer prognosis but also understanding the fundamental mechanism of mtp53 oncogenic action. Here we assessed the influence of mtp53 in breast cancers to the mechanical property of breast cancer cells.

Adherence to psychostimulant medication in children with attention-deficit/hyperactivity disorder: the role of attitudes.

Objective: To investigate how attitudes towards psychostimulant medication influence the adherence to psychostimulant treatment in children with Attention-Deficit/Hyperactivity Disorder (ADHD).

Method: Thirty-three children with ADHD were prospectively recruited to take part in this study. The children and their parents filled questionnaires at both baseline and at a three-month follow-up to assess the severity of ADHD symptoms in the child and attitudes towards psychostimulant medication.

Endogenous human MDM2-C is highly expressed in human cancers and functions as a p53-independent growth activator.

Human cancers over-expressing mdm2, through a T to G variation at a single nucleotide polymorphism at position 309 (mdm2 SNP309), have functionally inactivated p53 that is not effectively degraded. They also have high expression of the alternatively spliced transcript, mdm2-C. Alternatively spliced mdm2 transcripts are expressed in many forms of human cancer and when they are exogenously expressed they transform human cells.

Efficacy of Methylphenidate in ADHD Children across the Normal and the Gifted Intellectual Spectrum.

Objective: This study evaluates whether attention-deficit/hyperactivity disorder (ADHD) children with a borderline intelligence quotient (IQ) (70≤FSIQ<80), normal IQ (80≤FSIQ<120) and high IQ (FSIQ≥120) respond differently to psychostimulant treatment.

Method: 502 children, aged 6 to 12 years, with an IQ range from 70 to 150 participated in a two-week, double-blind, placebo-controlled, crossover methylphenidate (MPH) trial.

Results: In addition to differences in socioeconomic background and parental education, higher IQ children were found to present with less severe symptoms.

8-Amino-adenosine activates p53-independent cell death of metastatic breast cancers.

8-Amino-adenosine (8-NH(2)-Ado) is a ribose sugar nucleoside analogue that reduces cellular ATP levels and inhibits mRNA synthesis. Estrogen receptor-negative (ER-) metastatic breast cancers often contain mutant p53; therefore, we asked if 8-NH(2)-Ado could kill breast cancer cells without activating the p53-pathway. Regardless of the breast cancer subtype tested or the p53 status of the cells, 8-NH(2)-Ado was more cytotoxic than either gemcitabine or etoposide.

Mutant p53 disrupts mammary tissue architecture via the mevalonate pathway.

p53 is a frequent target for mutation in human tumors, and mutant p53 proteins can actively contribute to tumorigenesis. We employed a three-dimensional culture model in which nonmalignant breast epithelial cells form spheroids reminiscent of acinar structures found in vivo, whereas breast cancer cells display highly disorganized morphology. We found that mutant p53 depletion is sufficient to phenotypically revert breast cancer cells to a more acinar-like morphology.

Relation between therapeutic response and side effects induced by methylphenidate as observed by parents and teachers of children with ADHD.

Background: The desired (therapeutic) and undesired (side) effects of methylphenidate might have underlying correlations. The aim of this study was to explore the strength and the possible sources of these correlations.

Methods: One hundred and fifty-seven children with ADHD (6-12 years) were administered placebo and methylphenidate (0.

A p53-independent role of Mdm2 in estrogen-mediated activation of breast cancer cell proliferation.

Introduction: Estrogen receptor positive breast cancers often have high levels of Mdm2. We investigated if estrogen signaling in such breast cancers occurred through an Mdm2 mediated pathway with subsequent inactivation of p53.

Methods: We examined the effect of long-term 17β-estradiol (E2) treatment (five days) on the p53-Mdm2 pathway in estrogen receptor alpha (ERα) positive breast cancer cell lines that contain wild-type p53 (MCF-7 and ZR75-1).

COMT Val108/158Met polymorphism and the modulation of task-oriented behavior in children with ADHD.

It has been suggested that the symptoms of attention-deficit/hyperactivity disorder (ADHD), including inattention and/or hyperactivity/impulsivity, translate into deficits in task-oriented behavior or problem-focused activity. The frontosubcortical dopamine pathway has been implicated in ADHD. One of the key modulators of extracellular dopamine levels in the prefrontal cortex is catechol-O-methyltransferase (COMT).

Dopamine transporter 3'UTR VNTR genotype is a marker of performance on executive function tasks in children with ADHD.

Background: Attention-Deficit/Hyperactivity Disorder (ADHD) is a heterogeneous disorder from both clinical and pathogenic viewpoints. Executive function deficits are considered among the most important pathogenic pathways leading to ADHD and may index part of the heterogeneity in this disorder.

Methods: To investigate the relationship between the dopamine transporter gene (SLC6A3) 3'-UTR VNTR genotypes and executive function in children with ADHD, 196 children diagnosed with ADHD were sequentially recruited, genotyped, and tested using a battery of three neuropsychological tests aimed at assessing the different aspects of executive functioning.

Lipid mediators of autophagy in stress-induced premature senescence of endothelial cells.

Our group (Patschan S, Chen J, Gealekman O, Krupincza K, Wang M, Shu L, Shayman JA, Goligorsky MS; Am J Physiol Renal Physiol 294: F100-F109, 2008) previously observed an accumulation of gangliosides coincident with development of cell senescence and demonstrated lysosomal permeabilization in human umbilical vein endothelial cells exposed to glycated collagen I (GC). Therefore, we investigated whether the lysosome-dependent, caspase-independent or type 2-programmed cell death (autophagy) is involved in development of premature senescence of endothelial cells. The cleaved microtubule-associated protein 1 light-chain 3 (LC3), a marker of autophagosome formation, was overexpressed within 24 h of GC treatment; however, by 4-5 days, it was nearly undetectable.

Relation of maternal stress during pregnancy to symptom severity and response to treatment in children with ADHD.

Objective: There is considerable evidence that maternal stress is associated with behavioural disturbances in offspring. The objective of this study was to examine whether there is an association between the severity of maternal stress during pregnancy and the severity of symptoms of attention-deficit hyperactivity disorder (ADHD). A second objective was to examine whether there is an association between maternal stress and children's response to methylphenidate (MPH).

COMT Val108/158Met gene variant, birth weight, and conduct disorder in children with ADHD.

Objective: In a recent study, Thapar and colleagues reported that COMT "gene variant and birth weight predict early-onset antisocial behavior in children" with attention-deficit/hyperactivity disorder. We have attempted to replicate these findings in a group of ADHD children using a similar research design.

Method: Children (n=191) between 6 and 12 years of age who were diagnosed with ADHD were included in the study.

[Signaling pathways that control the growth and survival of prostate tumor cells in the absence of androgens].

Androgen-dependent human prostate adenocarcinoma cell line LNCaP was used to study the effect of androgen deprivation on the cell response to TNF-related cytokines. Several signaling pathways were implicated in cell survival in the absence of androgens. In androgen-deprived LNCaP cells, TNF-alpha and TRAIL stimulated the cell growth and activated the mitogenic and antiapoptotic signaling pathways involving NF-kappa B, STAT3, PI3K, and beta-catenin.

[Differences in expression and functional organization of the rat tyrosine aminotransferase gene in two lines of Morris hepatoma, 8994 and 7777].

Expression and structural organization of tyrosine aminotransferase (TAT) gene in Morris hepatoma cell line 7777 with active and glucocorticoid-inducible TAT gene and in hepatoma 8994, where TAT gene does not function were analysed. No differences in the number of receptor macromolecules, translocation and nuclear binding of hormone-receptor complexes in hormone sensitive (7777) and resistant (8994) cell lines were demonstrated. Dexamethasone increases TAT gene transcription in 7777 cell line but not 8994.

[Activation of transcription of the tyrosine aminotransferase gene in the rat McA-RN 7777 hepatoma cell line by cycloheximide].

The expression of the tyrosine aminotransferase (TAT) mRNA after cycloheximide treatment was analysed by Northern blotting method in Morris rat hepatoma cell lines. The level of TAT mRNA increased after 6-8 h of cycloheximide treatment only in the McA-RH 7777 cell line. McA-RH 7777 nuclear run-off assay showed that TAT transcription was induced by cycloheximide treatment.

[Regulation of proliferative and protein-synthesizing reactions in human peripheral blood lymphocytes by glucocorticoids].

Glucocorticoids control protein synthesis and [3H]thymidine incorporation into DNA in resting and mitogen-stimulated in vitro human peripheral blood lymphocytes. This effect depends on the dose and time of hormone addition to the culture medium. Using two-dimensional electrophoresis, it was demonstrated that the steroids can regulate the expression of various proteins in intact and mitogen-stimulated human peripheral blood lymphocytes.

[Nuclear factor A5 from the rat liver that requires a metal for specific interaction in vitro with distal element of the tyrosine aminotransferase gene promoter].

To detect nuclear protein factors which might account for a tissue-specific and inducible expression of the rat tyrosine aminotransferase (TAT) gene promoter, extracts from rat liver and spleen nuclei have been fractionated by heparin-sepharose chromatography and the fractions assayed for sequence-specific binding to the distal TAT gene promoter element (sequence between -313 and -210). Gel retardation experiments carried out in the presence or absence of Mg2+, Ca2+, or Zn2+ ions showed that there are at least two nuclear factors (A3 and A4) binding to the distal promoter element only in the presence of the chelator (20 mM EDTA). Incubation of the protein fractions with Zn2+ or Ca2+ instead of commonly used Mg2+allowed: (i) to avoid 3 2P-DNA-probe degradation by "contaminating" endogenous nucleases; and (ii) to detect another sequence-specific nuclear factor, A5.