Biomedical subjects

Linda H Malkas

Publications and source records attributed to Linda H Malkas.

17 recordsLinked to original sources

PubMed2006

A cancer-associated PCNA expressed in breast cancer has implications as a potential biomarker.

Two isoforms of proliferating cell nuclear antigen (PCNA) have been observed in breast cancer cells. Commercially available antibodies to PCNA recognize both isoforms and, therefore, cannot differentiate between the PCNA isoforms in malignant and nonmalignant breast epithelial cells and tissues. We have developed a unique antibody that specifically detects a PCNA isoform (caPCNA) associated with breast cancer epithelial cells grown in culture and breast-tumor tissues. Immunostaining studies using this antibody suggest that the caPCNA isoform may be useful as a marker of breast cancer and that the caPCNA-specific antibody could potentially serve as a highly effective detector of malignancy. We also report here that the caPCNA isoform functions in breast cancer-cell DNA replication and interacts with DNA polymerase delta. Our studies indicate that the caPCNA isoform may be a previously uncharacterized detector of breast cancer.

Antibodies↗

PubMed2006

A novel biomarker for the detection of esophageal adenocarcinoma.

OBJECTIVES: Proliferating cell nuclear antigen is a component of the DNA synthesome and functions in DNA replication and repair. Our group has recently identified an acidic isoform of proliferating cell nuclear antigen, cancer-specific proliferating cell nuclear antigen, that appears to be present only in malignant tissue. We sought to determine the presence of cancer-specific proliferating cell nuclear antigen in esophageal dysplasias and invasive adenocarcinomas to assess its potential utility in discriminating malignancy. METHODS: With a polyclonal antibody to cancer-specific proliferating cell nuclear antigen, immunohistochemical staining was performed on samples from a total of 30 patients with Barrett esophagus with varying degrees of dysplasia and 18 patients with invasive adenocarcinoma. We also performed cancer-specific proliferating cell nuclear antigen immunohistochemical staining on a commercially available tissue microarray and on specimens obtained from endoscopic biopsies. As controls, immunohistochemical staining for cancer-specific proliferating cell nuclear antigen was performed on normal esophageal tissue and immunohistochemical staining for proliferating cell nuclear antigen was performed on all specimens with a commercially available antibody. RESULTS: Of the Barrett esophagus specimen, 14 showed no dysplasia, 8 showed low-grade dysplasia, and 8 showed high-grade dysplasia. None of these specimens stained positively for cancer-specific proliferating cell nuclear antigen. Of the 18 adenocarcinoma specimens, all stained positively for cancer-specific proliferating cell nuclear antigen. There was no significant cancer-specific proliferating cell nuclear antigen expression in normal esophageal tissue, and proliferating cell nuclear antigen expression was noted to a high degree in all tissues. CONCLUSIONS: Cancer-specific proliferating cell nuclear antigen appears to demonstrate high specificity for esophageal adenocarcinoma. This marker therefore may prove useful in differentiating invasive cancer from high-grade dysplasia. Cancer-specific proliferating cell nuclear antigen also holds future promise as a biomarker for esophageal adenocarcinoma.

Adenocarcinoma↗

PubMed2006

Proteomic analysis of neuroblastoma subtypes in response to mitogen-activated protein kinase inhibition: profiling multiple targets of cancer kinase signaling.

INTRODUCTION: Survival for high-risk neuroblastoma (NB) remains poor despite aggressive therapy. Novel therapies are vital for improving prognosis. We previously showed differential NB subtype sensitivity to p42/44 mitogen-activated protein kinase (ERK/MAPK) pathway inhibition. In this study, we investigated proteomic changes associated with resistance or sensitivity to MAPK kinase (MEK) inhibition in NB subtypes. MATERIALS AND METHODS: SH-SY5Y (N-type), BE(2)-C (I-type), and SK-N-AS (S-type) were treated with MEK inhibitor U0126 (10 microM) for 1 and 24 h. Proteins were extracted from untreated and treated cells and analyzed for differential expression by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Selected polypeptides were extracted from the gel and identified by liquid chromatography-linked tandem mass spectrometry (LC-MS/MS). RESULTS: We identified 15 proteins that were decreased by 2.5-fold between untreated and 1 h treated cells and subsequently up-regulated 5-fold after 24 h drug treatment. N-type NB (MEK-resistant) showed the least altered proteomic profile whereas the I-type (MEK-sensitive) and S-type NB (MEK-intermediate) generated significant protein changes. The majority of proteins identified were induced by stress. CONCLUSIONS: Protein differences exist between MEK inhibitor-treated NB subtypes. Identified polypeptides all have roles in mediating cellular stress. These data suggest that inhibition of the ERK/MAPK in NB subtypes leads to an intracellular stress response. The most resistant NB cell line to MEK inhibitor treatment generated the least protective protein profile, whereas the intermediate and most sensitive NB cells produced the most stress response. These findings suggest stress related protein expression may be targeted in assessing a response to ERK/MAPK therapeutics.

Butadienes↗

PubMed2006

The discovery of labile methyl esters on proliferating cell nuclear antigen by MS/MS.

The post-translational modification of proliferating cell nuclear antigen (PCNA) has been implicated in modulating its function for over 20 years. With multiple interacting partners, PCNA is involved in processes ranging from DNA replication and repair to cell cycle control and apoptosis. The ability of PCNA to distinguish between specific binding partners in different tasks is currently of intense interest, and several post-translational modifications have been reported to modulate its function. Unfortunately, these reports have produced contradictory information on the type(s) of modification present on the molecule. Here we report a detailed structural analysis of a single acidic PCNA isoform, cancer-specific polyferating nuclear anitgen (csPCNA), isolated from breast cancer cells by 2D-PAGE and LC-MS/MS. With this approach we fully characterized the csPCNA isoform and confidently identified a single post-translational modification, methyl esterification. Interestingly, the methyl esters consistently localized to 15 specific glutamic and aspartic acid residues of csPCNA. The methyl esterification of csPCNA represents a novel type of post-translational modification in mammalian cells that could ultimately hold the key towards unlocking its diverse functions.

Amino Acid Sequence↗

PubMed2006

Structural analysis of the human neuroblastoma DNA replication complex: insights into faulty proliferation.

BACKGROUND: Whether faulty DNA synthesis contributes to neuroblastoma (NB) pathogenesis has received little attention. We have shown that NB DNA replication is orchestrated by a multiprotein DNA replication complex (DNA synthesome) which mediates error-prone DNA synthesis. We sought to define proteomic alterations of the NB DNA synthesome, which lead to lowered replication fidelity. METHODS: DNA synthesomes from NB and neural stem (NS) cell culture were purified. Proteomic differences of the DNA synthesome between NB and NS cells were determined using 2-dimensional polyacrylamide gel electrophoresis and immunodetection. RESULTS: DNA synthesome proteins from NB and NS cells were compared. Only replication protein A (RPA) showed distinct changes. RPA from NS cells resolved as a single spot (isoelectric point [pI] 5.75), whereas RPA in NB showed a main spot (pI 5.75) along with 3 additional isoforms. RPA binds to single-stranded DNA and participates in protein-protein interactions. CONCLUSIONS: NB DNA synthesome showed 3 distinct isoforms of RPA when compared with NS cells. These findings are significant in that it is possible to link changes in the fidelity of DNA replication with a specific protein alteration of the NB DNA synthetic apparatus. The novel RPA forms may be a new signature of NB malignancy.

Blotting, Western↗

PubMed2006

Differential sensitivity of chemoresistant neuroblastoma subtypes to MAPK-targeted treatment correlates with ERK, p53 expression, and signaling response to U0126.

PURPOSE: Neuroblastoma tumors are comprised of neuroblastic (N), substrate-adherent (S), and intermediate (I) cells. Because cell growth and differentiation often involve p44/p42 mitogen-activated protein kinase (MAPK) pathway signaling, we explored MAPK signaling and growth response in three NB cell types after MAPK kinase (MEK) inhibition to evaluate the feasibility of MAPK-targeted treatment strategies. METHODS: Three human NB cell cultures, SH-SY5Y (N-type), BE(2)-C (I-type), and SK-N-AS (S-type), were treated in monolayer cultures with increasing concentrations of the MEK inhibitor U0126. MAPK pathway intermediates MEK and ERK, their activated (phosphorylated) forms p-MEK and p-ERK, and p53 expression were assessed by Western blot at 1 and 24 hours. At 72 hours, cell counts determined growth inhibition and DNA fragmentation ELISA assessed apoptosis. RESULTS: Among all three lines, total ERK and MEK expression were unaffected by U0126. However, constitutive total ERK and p53 expression were significantly greater in BE(2)-C (I-type) cells than in SH-SY5Y (N-type) and SK-N-AS (S-type). Active ERK (p-ERK) levels decreased in dose response to U0126 at 1 and 24 hours in all lines. Conversely, p-MEK levels increased with increasing U0126 concentrations at 1 hour in SH-SY5Y (N-type) and at 24 hours in all lines. BE(2)-C (I-type) cell counts decreased in concentration-dependent fashion with U0126, whereas SH-SY5Y (N-type) and SK-N-AS (S-type) showed a biphasic response with increased cell counts at 1 micromol/L U0126 and slightly decreased cell counts at 10 mumol/L U0126. CONCLUSION: This study demonstrates that BE(2)-C (I-type) cells exhibit greater constitutive total ERK and p53 expression than SH-SY5Y (N-type) and SK-N-AS (S-type). Although all three lines exhibit p-ERK decreases with MEK inhibition, only BE(2)-C (I-type) cells significantly decrease their proliferation with U0126 treatment. Although MEK inhibition holds promise in targeting I-type NB cells, successfully treating this heterogeneous tumor may require combining agents against N- and S-type cells.

Apoptosis↗

PubMed2006

Novel peptides secreted from human neuroblastoma: useful clinical tools?

BACKGROUND: Differentially expressed neuroblastoma (NB) proteins are vital for the development of new diagnostics and therapeutics. For example, secretory NB peptides (neuron-specific enolase and chromogranins) are clinically useful. We investigated polypeptide secretion by employing proteomic technologies to analyze proteins released from cultured NB cells. METHODS: Neuroblastoma cell lines (SK-N-AS, SK-N-DZ, and SK-N-FI) were grown in serum-free media. Conditioned media from each cell line was analyzed for secreted proteins by 2-dimensional polyacrylamide gel electrophoresis. Selected polypeptides were identified by liquid chromatography-linked tandem mass spectrometry. RESULTS: We identified 5 polypeptides that were secreted or shed by NB. Ubiquitin, beta2-microglobulin, insulin-like growth factor binding protein-2, superoxide dismutase (copper and zinc), and heat shock cognate 70-kd proteins were secreted from NB cells, as compared with control media. Elevated levels of these proteins have been described in serum/tissues under intracellular stress and malignancies, including NB. CONCLUSION: These novel secretory polypeptides may contribute to NB growth. The proteins may reveal additional tumor markers and permit putative use in the diagnosis and treatment of NB. Detection of these proteins in serum of children with NB vs controls (using 2-dimensional polyacrylamide gel electrophoresis and mass spectrometry techniques) is currently in progress.

Biomarkers, Tumor↗

PubMed2006

Neuroblastoma detection using serum proteomic profiling: a novel mining technique for cancer?

BACKGROUND: Serum proteins in neuroblastoma (NB), such as neuron-specific enolase and lactate dehydrogenase, are used as nonspecific markers of disease severity. In this study, we have generated serum protein profiles that correlate with NB by applying proteomic technologies to uncover, at the protein level, serum polypeptide expression patterns in patients with NB. METHODS: Surface-enhanced laser desorption/ionization-time-of-flight mass spectrometry was used to generate protein expression spectra in human NB (group I, n = 18) and healthy children (group II, n = 17) sera. Groups I and II mass spectral data were compared after baseline subtraction. Peaks with high signal-to-noise ratios were selected and grouped into bins with various intervals along mass-to-charge axis. Two-sample t test and 3-fold cross validation were used to identify differential biomarkers between groups I and II. RESULTS: Significant differentially expressed proteins were identified between groups I and II (P < or = .05). The discriminatory features (proteomic patterns) of cancer from normal sera were successfully identified using the classification algorithm. The average classification performance after 3-fold cross validation was 87.26%. CONCLUSION: Surface-enhanced laser desorption/ionization-time-of-flight mass spectrometry is suitable for preliminary assessment of NB and could potentially provide a noninvasive diagnosis of NB. We propose that surface-enhanced laser desorption/ionization provides a novel method for NB diagnosis because direct observations of spectral differences between normal and NB sera are possible.

Child, Preschool↗

PubMed2005

DNA damage-induced phosphorylation of the human telomere-associated protein TRF2.

Several protein kinases from diverse eukaryotes known to perform important roles in DNA repair have also been shown to play critical roles in telomere maintenance. Here, we report that the human telomere-associated protein TRF2 is rapidly phosphorylated in response to DNA damage. We find that the phosphorylated form of TRF2 is not bound to telomeric DNA, as is the ground form of TRF2, and is rapidly localized to damage sites. Our results suggest that the ataxia-telangiectasia-mutated (ATM) protein kinase signal-transduction pathway is primarily responsible for the DNA damage-induced phosphorylation of TRF2. Unlike DNA damage-induced phosphorylation of other ATM targets, the phosphorylated form of TRF2 is transient, being detected rapidly at DNA damage sites postirradiation, but largely dissipated by 2 hours. In addition, we report that the phosphorylated form of TRF2 is present at telomeres in cell types undergoing telomere-based crisis and a recombination-driven, telomerase-independent, alternative lengthening of telomeres (ALT) pathway, likely as a consequence of a telomere-based DNA damage response. Our results link the induction of TRF2 phosphorylation to the DNA damage-response system, providing an example of direct cross-talk via a signaling pathway between these two major cellular processes essential for genomic stability, telomere maintenance, and DNA repair.

Amino Acid Sequence↗

PubMed2005

Profiling of nuclear extract proteins from human neuroblastoma cell lines: the search for fingerprints.

PURPOSE: Neuroblastoma (NB) commonly presents with advanced disease at diagnosis and is associated with poor survival. If identified early, however, survival is improved suggesting a benefit of early detection. The authors have used proteomics technology in an attempt to identify novel markers that permit early detection of NB and characterize its molecular makeup. METHODS: Three different human NB cell lines SK-N-AS, SK-N-DZ, and SK-N-FI were subjected to series of biochemical fractionation steps to extract nuclear proteins. These proteins were analyzed for differential expression by 2-dimensional polyacrylamide gel electrophoresis. Polypeptides of interest were subsequently identified by liquid chromatography-linked tandem mass spectrometry. RESULTS: Multiple proteins were identified in these human NB cell lines including SET (a ubiquitous nuclear protein), stathmin (a cytosolic signal transduction protein), and grp94 (a heat shock protein). SET is a putative oncogene associated with the chromosomal translocation (6;9) leading to acute undifferentiated leukemia. Stathmin is an oncogene found in greater abundance in leukemic cells compared to nonleukemic cells. A total of 94-kDa glucose-regulated protein has been shown to be protective in human breast cancer cells in vitro and related with the occurrence, differentiation, and progression of human lung cancer. The first protein has not been previously associated with NB. CONCLUSIONS: The identification of these 3 previously unrecognized cancer-related potential biomarkers in human NB cell lines may prove useful in developing diagnostic tests. The proteomic methodology of 2-dimensional polyacrylamide gel electrophoresis/mass spectrometry also provides an improved opportunity to understand the natural history of NB and develop novel chemotherapeutic agents for this prevalent childhood malignancy with a dismal outcome.

Biomarkers, Tumor↗

PubMed2005

Isolation and characterization of a DNA synthesome from a neuroblastoma cell line.

BACKGROUND: Despite significant analysis of the chromosomal abnormalities associated with neuroblastoma (NB), the role that NB DNA replication may play in the accumulation of genetic damage is poorly understood. For that matter, the mechanisms involved in NB DNA synthesis have yet to be elucidated. In an effort to investigate this process in NB, we have isolated and purified a multiprotein DNA replication complex from human NB cells (IMR-32). METHODS: Using a series of subcellular fractionations, ion-exchange chromatography, and gradient sedimentation steps, we have isolated a simian virus 40 replication competent multiprotein complex from IMR-32 NB cells, which has been designated the DNA synthesome. Enzymatic and immunodetection techniques were used to characterize the multiple components of the multiprotein DNA replication complex. RESULTS: The NB DNA synthesome was found to remain intact and functional through all the steps of its purification. The proteins and enzymatic activities that were found to copurify with the NB DNA synthesome include: DNA polymerases alpha , delta , and epsilon , proliferating cell nuclear antigen, replication factor A, replication factor C, topoisomerases I and II, flap endonuclease 1, and DNA ligase I. CONCLUSION: Although the cooperative integration of a DNA replication macromolecular complex (DNA synthesome) is not new, we extend the view of the DNA synthesome mediating DNA synthesis for human NB. The data reported here characterize the human NB DNA synthesome for the first time and provide the groundwork for investigating whether the NB DNA synthesome contributes to faulty DNA replication and tumor pathogenesis for this childhood malignancy.

Chromatography, Ion Exchange↗

PubMed2004

Altered DNA polymerase iota expression in breast cancer cells leads to a reduction in DNA replication fidelity and a higher rate of mutagenesis.

The recently discovered human enzyme DNA polymerase iota (pol iota) has been shown to have an exceptionally high error rate on artificial DNA templates. Although there is a considerable body of in vitro evidence for a role for pol iota in DNA lesion bypass, there is no in vivo evidence to confirm this action. We report here that pol iota expression is elevated in breast cancer cells and correlates with a significant decrease in DNA replication fidelity. We also demonstrate that UV treatment of breast cancer cells additionally increases pol iota expression with a peak occurring between 30 min and 2 h after cellular insult. This implies that the change in pol iota expression is an early event after UV-mediated DNA damage. That pol iota may play a role in the higher mutation frequencies observed in breast cancer cells was suggested when a reduction in mutation frequency was found after pol iota was immunodepleted from nuclear extracts of the cells. Analysis of the UV-induced mutation spectra revealed that > 90% were point mutations. The analysis also demonstrated a decreased C --> T nucleotide transition and an increased C --> A transversion rate. Overall, our data strongly suggest that pol iota may be involved in the generation of both increased spontaneous and translesion mutations during DNA replication in breast cancer cells, thereby contributing to the accumulation of genetic damage.

Base Sequence↗

PubMed2004

An in vitro model system that can differentiate the stages of DNA replication affected by anticancer agents.

We have previously reported on the potential use of a novel in vitro human cell-derived model system to investigate the mechanism of action of anticancer agents that directly affect the process of DNA replication. Our cell-free system uses a multiprotein DNA replication complex (designated the DNA synthesome) that has been isolated, characterized, and extensively purified from a wide variety of mammalian cells and tissues. The DNA synthesome is competent to orchestrate simian virus 40 (SV40) origin-specific and large T antigen-dependent DNA replication in vitro. In this study, the synthesome-based cell-free system was tested to evaluate the mechanism of action of 1-beta-d-arabinofuranosylcytosine (ara-C), camptothecin (CPT), and doxorubicin (DOX). Using a novel synthesome-based in vitro kinetic assay, we demonstrated that DNA replication mediated by the synthesome is initiated within the SV40 replication origin and proceeds bidirectionally in a manner analogous to that occurring within the cell. Ara-CTP, CPT, and DOX have been found to affect different stages of the in vitro DNA replication process mediated by the complex. Ara-CTP inhibited both the initiation and elongation stages, whereas CPT produced most of its effects by inhibiting the elongation phase of DNA replication. DOX inhibited the termination stage of DNA synthesis mediated by the synthesome. The data presented here support our contention that the DNA synthesome represents a highly effective in vitro model system for investigating the mechanism by which some anticancer agents can directly affect the process of DNA replication.

Antineoplastic Agents↗

PubMed2003

The DNA synthesome: its potential as a novel in vitro model system for studying S-phase specific anticancer agents.

A considerable effort is being made to discover more effective anticancer agents and to develop rapid, accurate, and reliable methods for evaluating the usefulness of the new compounds and analyzing their mechanisms of action. In this regard, our laboratory was the first to report the isolation, characterization, and extensive purification of an intact, stable, and fully functional mammalian cell multiprotein complex, which we designated the DNA synthesome. The DNA synthesome has been isolated from a wide variety of mammalian cells as well as from tissues and has been shown to be fully competent to support SV40 origin-specific and large T antigen-dependent DNA replication in vitro. In this article we will review the utilization of the DNA synthesome as a powerful in vitro model system for analyzing the mechanism of action of S-phase specific anticancer agents.

Antineoplastic Agents↗

PubMed2002

Human cell DNA replication is mediated by a discrete multiprotein complex.

A discrete high molecular weight multiprotein complex containing DNA polymerase alpha has been identified by a native Western blotting technique. An enrichment of this complex was seen at each step in its purification. Further purification of this complex by ion-exchange chromatography indicates that the peak of DNA polymerase alpha activity co-purifies with the peak of in vitro SV40 DNA replication activity eluting from the column. The complex has a sedimentation coefficient of 18S in sucrose density gradients. We have designated this complex as the DNA synthesome. We further purified the DNA synthesome by electroeluting this complex from a native polyacrylamide gel. The eluted complex retains in vitro DNA synthetic activity, and by Western blot analysis, contains DNA polymerase delta, proliferating cell nuclear antigen, and replication protein A. Enzymatic analysis of the electroeluted DNA synthesome indicates that the synthesome contains topoisomerase I and II activities, and SDS-PAGE analysis of the electroeluted DNA synthesome revealed the presence of at least 25 major polypeptides with molecular weights ranging from 20 to 240 kDa. Taken together, our evidence suggests that the DNA synthesome may represent the minimal DNA replication unit of the human cell.

Cell Line↗

PubMed2002

Synthesis and preliminary pharmacological evaluation of new (+/-) 1,4-naphthoquinones structurally related to lapachol.

Seven new 1,4-naphthoquinones structurally related to lapachol were synthesized from lawsone and oxygenated arylmercurials. These compounds can also be seen as pterocarpan derivatives where the A-ring was substituted by the 1,4-naphthoquinone nucleus. Pharmacological screening provided evidence of significant biological activities, including effects against proliferation of the MCF-7 human breast cancer cell line, against Herpes Simplex Virus type 2 infection, and against snake poison-induced myotoxicity. One derivative displaced flunitrazepam binding and showed benzodiazepine-like activity, suggesting novel neuroactive structural motifs.

Animals↗

PubMedDate not supplied

DNMT1 is a component of a multiprotein DNA replication complex.

DNA methylation is a major determinant of epigenetic inheritance and plays an important role in genome stability. The accurate propagation of DNA methylation patterns with cell division requires that methylation be closely coupled to DNA replication, however the precise molecular determinants of this interaction have not been defined. In the present study, we show that the predominant DNA methyltransferase species in somatic cells, DNMT1, is a component of a multiprotein DNA replication complex termed the DNA synthesome that fully supports semi-conservative DNA replication in a cell-free system. DNMT1 protein and activity were found to co-purify with the human DNA synthesome through a series of subcellular fractionation and chromatography steps, resulting in an enrichment of methyltransferase specific activity from two human cell lines. DNA methyltransferase activity co-eluted with in vitro replication activity and DNA polymerase alpha activity on sucrose density gradients suggesting that DNMT1 is a tightly bound, core component of the replication complex. The synthesome-associated pool of DNA methyltransferase exhibited both maintenance and de novo methyltransferase activity and the ratio of the two was similar to that observed in whole cell lysates and for recombinant DNMT1. These data indicate that interactions within the synthesome complex do not influence the intrinsic preference of DNMT1 for hemimethylated DNA, but suggest that newly replicated DNA may be subject to low level de novo methylation. The data indicate that DNA methylation is tightly coupled to replication through physical interaction of DNMT1 and core components of the replication machinery. The definition of the molecular interactions between DNMT1 and other proteins in the replication complex in normal and neoplastic cells will provide further insight into the regulation of DNA methylation and the mechanisms underlying the alteration of DNA methylation patterns during carcinogenesis.

Cell Division↗