Phosphorylation of actopaxin regulates cell spreading and migration.

Actopaxin is an actin and paxillin binding protein that localizes to focal adhesions. It regulates cell spreading and is phosphorylated during mitosis. Herein, we identify a role for actopaxin phosphorylation in cell spreading and migration. Stable clones of U2OS cells expressing actopaxin wild-type (WT), nonphosphorylatable, and phosphomimetic mutants were developed to evaluate actopaxin function. All proteins targeted to focal adhesions, however the nonphosphorylatable mutant inhibited spreading whereas the phosphomimetic mutant cells spread more efficiently than WT cells. Endogenous and WT actopaxin, but not the nonphosphorylatable mutant, were phosphorylated in vivo during cell adhesion/spreading. Expression of the nonphosphorylatable actopaxin mutant significantly reduced cell migration, whereas expression of the phosphomimetic increased cell migration in scrape wound and Boyden chamber migration assays. In vitro kinase assays demonstrate that extracellular signal-regulated protein kinase phosphorylates actopaxin, and treatment of U2OS cells with the MEK1 inhibitor UO126 inhibited adhesion-induced phosphorylation of actopaxin and also inhibited cell migration.

GDF-3 is an adipogenic cytokine under high fat dietary condition.

Growth differentiation factor 3 (GDF-3) is structurally a bone morphogenetic protein/growth differentiation factor subfamily member of the TGF-beta superfamily. GDF-3 exhibits highest level of expression in white fat tissue in mice and is greatly induced by high fat diet if fat metabolic pathway is blocked. To identify its biological function, GDF-3 was overexpressed in mice by adenovirus mediated gene transfer. Mice transduced with GDF-3 displayed profound weight gain when fed with high fat diet. The phenotypes included greatly expanded adipose tissue mass, increased body adiposity, highly hypertrophic adipocytes, hepatic steatosis, and elevated plasma leptin. GDF-3 stimulated peroxisome proliferator activated receptor expression in adipocytes, a master nuclear receptor that controls adipogenesis. However, GDF-3 was not involved in blood glucose homeostasis or insulin resistance, a condition associated with obesity. In contrast, similar phenotypes were not observed in GDF-3 mice fed with normal chow, indicating that GDF-3 is only active under high lipid load. Thus, GDF-3 is a new non-diabetic adipogenic factor tightly coupled with fat metabolism.

Oncogenic K-ras drives cell cycle progression and phenotypic conversion of primary pancreatic duct epithelial cells.

We have established a primary pancreatic duct epithelial cell culture (PDEC) system to investigate the relationship between oncogenic activation of K-ras and pancreatic ductal tumorigenesis. We have found that the acute introduction of physiological levels of oncogenic K-ras (K-rasV12) into quiescent PDECs stimulates S-phase entry and induces a pronounced increase in cell size. Both effects are dependent on the functional integrity of the phosphatidylinositol 3'-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway. In addition, K-rasV12 promotes the loss of epithelial E-cadherin and the gain of mesenchymal N-cadherin in PDEC. Our observations indicate that the oncogenic activation of K-ras is sufficient to elicit mitogenic and morphogenic responses in pancreatic ductal cells and hence is likely to play an instructive role in the initiation of pancreatic ductal adenocarcinoma.

Proteomic, functional, and domain-based analysis of in vivo 14-3-3 binding proteins involved in cytoskeletal regulation and cellular organization.

BACKGROUND: 14-3-3 proteins are abundant and conserved polypeptides that mediate the cellular effects of basophilic protein kinases through their ability to bind specific peptide motifs phosphorylated on serine or threonine. RESULTS: We have used mass spectrometry to analyze proteins that associate with 14-3-3 isoforms in HEK293 cells. This identified 170 unique 14-3-3-associated proteins, which show only modest overlap with previous 14-3-3 binding partners isolated by affinity chromatography. To explore this large set of proteins, we developed a domain-based hierarchical clustering technique that distinguishes structurally and functionally related subsets of 14-3-3 target proteins. This analysis revealed a large group of 14-3-3 binding partners that regulate cytoskeletal architecture. Inhibition of 14-3-3 phosphoprotein recognition in vivo indicates the general importance of such interactions in cellular morphology and membrane dynamics. Using tandem proteomic and biochemical approaches, we identify a phospho-dependent 14-3-3 binding site on the A kinase anchoring protein (AKAP)-Lbc, a guanine nucleotide exchange factor (GEF) for the Rho GTPase. 14-3-3 binding to AKAP-Lbc, induced by PKA, suppresses Rho activation in vivo. CONCLUSION: 14-3-3 proteins can potentially engage around 0.6% of the human proteome. Domain-based clustering has identified specific subsets of 14-3-3 targets, including numerous proteins involved in the dynamic control of cell architecture. This notion has been validated by the broad inhibition of 14-3-3 phosphorylation-dependent binding in vivo and by the specific analysis of AKAP-Lbc, a RhoGEF that is controlled by its interaction with 14-3-3.

The coxsackie adenovirus receptor inhibits cancer cell migration.

The coxsackie and adenovirus receptor (CAR) is a key factor in adenoviral cancer gene therapy. Reduced expression of CAR during progression of prostate and bladder cancer has been reported. In embryonic development and tissue differentiation, CAR is also differentially expressed. This study suggests a role of CAR expression in cell adhesion and cell motility of human cancer cells. Stable CAR-expressing clones from E-cadherin-deficient A2780 ovarian and CaSki cervical cancer cells with originally low and high CAR expression levels, respectively, were established. CAR reexpression in otherwise singularly growing A2780 parental cells resulted in formation of cell-cell contacts and aggregation in cell clusters. CAR overexpression in cell adhesion-forming CaSki cells did not result in morphological changes. Migration of the A2780 CAR clones was strongly reduced as characterized by using spread-off assays. Using migration chambers, formation of satellite colonies was reduced by 97% in CAR-expressing A2780 cell clones and by 23% in CAR-expressing CaSki cell clones. Parental A2780 and CaSki cells selected for high migratory ability by using migration chambers expressed endogenous CAR on lower levels associated with lower adenoviral transduction efficiency. Our data suggest CAR as a new inhibitory factor for cancer cell migration.

Sympathetic cholinergic target innervation requires GDNF family receptor GFR alpha 2.

Many cholinergic parasympathetic and enteric neurons require neurturin signaling through GDNF family receptor GFRalpha2 for target innervation. Since a distinct minority of sympathetic neurons are cholinergic, we examined whether GFRalpha2 is important for their development. We detected GFRalpha2 in neonatal sympathetic cholinergic neurons and neurturin mRNA in their target tissues, sweat glands in footpads, and periosteum. Lack of GFRalpha2 in mice did not affect the number of sympathetic cholinergic neurons, but their soma size was decreased in comparison to wild types. In adult and in 3-week-old GFRalpha2 knockout mice, the density of sympathetic cholinergic innervation was reduced by 50-70% in the sweat glands, and was completely absent in the periosteum. Sympathetic noradrenergic innervation of blood vessels in the footpads was unchanged. The density of sympathetic axons in sweat glands was unaffected at postnatal day P4 reflecting successful growth into the target area. Our results indicate that the cholinergic subpopulation of sympathetic neurons requires GFRalpha2 signaling for soma size and for growth or maintenance of target innervation. Thus, neurturin may be a general target-derived innervation factor for postganglionic cholinergic neurons in all parts of the autonomic nervous system.

Transgenic mice expressing the human C99 terminal fragment of betaAPP: effects on cytochrome oxidase activity in skeletal muscle and brain.

In order to furnish a combined model of relevance to human inclusion-body myopathy and Alzheimer's disease, transgenic mice expressing human betaAPP-C99 in skeletal muscle and brain under the control of the cytomegalovirus/beta-actin promoter were produced (Tg13592). These transgenic mice develop Abeta deposits in muscles but not in brain. Cell metabolic activity was analyzed in brain regions and muscle by cytochrome oxidase (CO) histochemistry, the terminal enzyme of the electron transport chain. By comparison to age-matched controls of the C57BL/6 strain, CO activity was selectively increased in dark skeletal muscle fibers of Tg13592 mice. In addition, only increases in CO activity were obtained in those brain regions where a significant difference appeared. The CO activity of Tg13592 mice was elevated in several thalamic nuclei, including laterodorsal, ventromedial, and midline as well as submedial, intralaminar, and reticular. In contrast, the groups did not differ in most cortical regions, except for prefrontal, secondary motor, and auditory cortices, and in most brainstem regions, except for cerebellar (fastigial and interpositus) nuclei and related areas (red and lateral vestibular nuclei). No variation in cell density and surface area appeared in conjunction with these enzymatic alterations. The overproduction of betaAPP-C99 fragments in brain without (amyloidosis did not appear to affect the metabolic activity of structures particularly vulnerable in Alzheimer's disease.

CDK activity antagonizes Whi5, an inhibitor of G1/S transcription in yeast.

Cyclin-dependent kinase (CDK) activity initiates the eukaryotic cell division cycle by turning on a suite of gene expression in late G1 phase. In metazoans, CDK-dependent phosphorylation of the retinoblastoma tumor suppressor protein (Rb) alleviates repression of E2F and thereby activates G1/S transcription. However, in yeast, an analogous G1 phase target of CDK activity has remained elusive. Here we show that the cell size regulator Whi5 inhibits G1/S transcription and that this inhibition is relieved by CDK-mediated phosphorylation. Deletion of WHI5 bypasses the requirement for upstream activators of the G1/S transcription factors SBF/MBF and thereby accelerates the G1/S transition. Whi5 is recruited to G1/S promoter elements via its interaction with SBF/MBF in vivo and in vitro. In late G1 phase, CDK-dependent phosphorylation dissociates Whi5 from SBF and drives Whi5 out of the nucleus. Elimination of CDK activity at the end of mitosis allows Whi5 to reenter the nucleus to again repress G1/S transcription. These findings harmonize G1/S control in eukaryotes.

Transgenic mouse in vivo library of human Down syndrome critical region 1: association between DYRK1A overexpression, brain development abnormalities, and cell cycle protein alteration.

Down syndrome is the most frequent genetic cause of mental retardation, having an incidence of 1 in 700 live births. In the present study we used a transgenic mouse in vivo library consisting of 4 yeast artificial chromosome (YAC) transgenic mouse lines, each bearing a different fragment of the Down syndrome critical region 1 (DCR-1), implicated in brain abnormalities characterizing this pathology. The 152F7 fragment, in addition to genes also located on the other DCR-1 fragments, bears the DYRK1A gene, encoding for a serine-threonine kinase. The neurobehavioral analysis of these mouse lines showed that DYRK1A overexpressing 152F7 mice but not the other lines display learning impairment and hyperactivity during development. Additionally, 152F7 mice display increased brain weight and neuronal size. At a biochemical level we found DYRK1A overexpression associated with a development-dependent increase in phosphorylation of the transcription factor FKHR and with high levels of cyclin B1, suggesting for the first time in vivo a correlation between DYRK1A overexpression and cell cycle protein alteration. In addition, we found an altered phosphorylation of transcription factors of CREB family. Our findings support a role of DYRK1A overexpression in the neuronal abnormalities seen in Down syndrome and suggest that this pathology is linked to altered levels of proteins involved in the regulation of cell cycle.

Loss of tuberous sclerosis complex 1 (Tsc1) expression results in increased Rheb/S6K pathway signaling important for astrocyte cell size regulation.

Individuals with tuberous sclerosis complex (TSC) develop central nervous system abnormalities that may reflect astrocyte dysfunction. In an effort to model astrocyte dysfunction in TSC, we generated mice lacking Tsc1 (hamartin) expression in astrocytes and demonstrated that Tsc1-null astrocytes exhibit abnormalities in contact inhibition growth arrest. In this study, we demonstrate that hamartin-deficient astrocytes are also defective in cell size regulation. We show that the increase in Tsc1-null astrocyte size is associated with increased activation of the S6-kinase pathway. In keeping with recent reports that the hamartin/tuberin complex may regulate Rheb and downstream S6K activation, we demonstrate that expression of either Rheb or S6K in primary astrocytes results in increased S6 pathway activation, and that inhibition of Rheb activity in Tsc1-deficient astrocytes using either pharmacologic or genetic strategies markedly reduces S6 activation. Collectively, these observations suggest that TSC inactivation in astrocytes results in defective cell size regulation associated with dysregulated Rheb/mTOR/S6K pathway activity.

Calcineurin initiates smooth muscle differentiation in neural crest stem cells.

The process of vascular smooth muscle cell (vSMC) differentiation is critical to embryonic angiogenesis. However, despite its importance, the vSMC differentiation program remains largely undefined. Murine gene disruption studies have identified several gene products that are necessary for vSMC differentiation, but these methodologies cannot establish whether or not a factor is sufficient to initiate the differentiation program. A gain-of-function system consisting of normal vSMC progenitor cells would serve as a useful complement to whole animal loss-of-function studies. We use such a system here, namely freshly isolated rat neural crest stem cells (NCSCs), to show that activation of the calcineurin signaling pathway is sufficient to drive these cells toward a smooth muscle fate. In addition, we present data suggesting that transforming growth factor (TGF)-beta1, which also causes NCSCs to differentiate into smooth muscle, activates calcineurin signaling in NCSCs, leading to a model in which activation of calcineurin signaling is the mechanism by which TGF-beta1 causes SMC differentiation in these cells.

The role of IL-5 for mature B-1 cells in homeostatic proliferation, cell survival, and Ig production.

B-1 cells, distinguishable from conventional B-2 cells by their cell surface marker, anatomical location, and self-replenishing activity, play an important role in innate immune responses. B-1 cells constitutively express the IL-5R alpha-chain (IL-5Ralpha) and give rise to Ab-producing cells in response to various stimuli, including IL-5 and LPS. Here we report that the IL-5/IL-5R system plays an important role in maintaining the number and the cell size as well as the functions of mature B-1 cells. The administration of anti-IL-5 mAb into wild-type mice, T cell-depleted mice, or mast cell-depleted mice resulted in reduction in the total number and cell size of B-1 cells to an extent similar to that of IL-5Ralpha-deficient (IL-5Ralpha(-/-)) mice. Cell transfer experiments have demonstrated that B-1 cell survival in wild-type mice and homeostatic proliferation in recombination-activating gene 2-deficient mice are impaired in the absence of IL-5Ralpha. IL-5 stimulation of wild-type B-1 cells, but not IL-5Ralpha(-/-) B-1 cells, enhances CD40 expression and augments IgM and IgG production after stimulation with anti-CD40 mAb. Enhanced IgA production in feces induced by the oral administration of LPS was not observed in IL-5Ralpha(-/-) mice. Our results illuminate the role of IL-5 in the homeostatic proliferation and survival of mature B-1 cells and in IgA production in the mucosal tissues.

Cell cycle progression in G1 and S phases is CCR4 dependent following ionizing radiation or replication stress in Saccharomyces cerevisiae.

To identify new nonessential genes that affect genome integrity, we completed a screening for diploid mutant Saccharomyces cerevisiae strains that are sensitive to ionizing radiation (IR) and found 62 new genes that confer resistance. Along with those previously reported (Bennett et al., Nat. Genet. 29:426-434, 2001), these genes bring to 169 the total number of new IR resistance genes identified. Through the use of existing genetic and proteomic databases, many of these genes were found to interact in a damage response network with the transcription factor Ccr4, a core component of the CCR4-NOT and RNA polymerase-associated factor 1 (PAF1)-CDC73 transcription complexes. Deletions of individual members of these two complexes render cells sensitive to the lethal effects of IR as diploids, but not as haploids, indicating that the diploid G1 cell population is radiosensitive. Consistent with a role in G1, diploid ccr4Delta cells irradiated in G1 show enhanced lethality compared to cells exposed as a synchronous G2 population. In addition, a prolonged RAD9-dependent G1 arrest occurred following IR of ccr4Delta cells and CCR4 is a member of the RAD9 epistasis group, thus confirming a role for CCR4 in checkpoint control. Moreover, ccr4Delta cells that transit S phase in the presence of the replication inhibitor hydroxyurea (HU) undergo prolonged cell cycle arrest at G2 followed by cellular lysis. This S-phase replication defect is separate from that seen for rad52 mutants, since rad52Delta ccr4Delta cells show increased sensitivity to HU compared to rad52Delta or ccr4Delta mutants alone. These results indicate that cell cycle transition through G1 and S phases is CCR4 dependent following radiation or replication stress.

Cytologic characteristics of pulmonary papillary adenoma. A case report.

BACKGROUND: Pulmonary papillary adenoma is a benign pulmonary neoplasm. Previously pulmonary papillary adenoma was described in terms of immunohistochemistry and ultrastructure. However, there are no previous reports describing the cytologic characteristics of pulmonary papillary adenoma. CASE: A 50-year-old male was admitted for evaluation of a coin lesion in the left upper lung field. Radiologic images showed a solid, round tumor approximately 25 mm in diameter in the left upper lung. Transbronchial needle aspiration biopsy (TBNA) was performed, and small numbers of atypical cells were collected. Adenocarcinoma was suggested clinically, and left upper segmentectomy was performed. The histologic diagnosis was pulmonary papillary adenoma. Imprint cytology of the cut surface of the tumor showed tumor cells arranged in sheets that contained scant or vesicular cytoplasm. The nuclei were oval or round, without obvious anisokaryosis, and their chromatin was fine, without hyperchromasia. Cytologically, the nuclei of the tumor cells in the imprint specimen (38.70 +/- 8.69 microns 2) were uniform in size and similar to the atypical cells in the TBNA specimen (38.29 +/- 11.56 microns 2) but significantly larger than the nuclei of the bronchial cells (23.61 +/- 5.98 microns 2) (P < .0001). CONCLUSION: The cytologic appearance of pulmonary papillary adenoma was characterized morphologically and morphometrically. The possibility of cytodiagnosis by TBNA was suggested.

Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment.

Commitment of stem cells to different lineages is regulated by many cues in the local tissue microenvironment. Here we demonstrate that cell shape regulates commitment of human mesenchymal stem cells (hMSCs) to adipocyte or osteoblast fate. hMSCs allowed to adhere, flatten, and spread underwent osteogenesis, while unspread, round cells became adipocytes. Cell shape regulated the switch in lineage commitment by modulating endogenous RhoA activity. Expressing dominant-negative RhoA committed hMSCs to become adipocytes, while constitutively active RhoA caused osteogenesis. However, the RhoA-mediated adipogenesis or osteogenesis was conditional on a round or spread shape, respectively, while constitutive activation of the RhoA effector, ROCK, induced osteogenesis independent of cell shape. This RhoA-ROCK commitment signal required actin-myosin-generated tension. These studies demonstrate that mechanical cues experienced in developmental and adult contexts, embodied by cell shape, cytoskeletal tension, and RhoA signaling, are integral to the commitment of stem cell fate.

Comparative proteomics of primitive hematopoietic cell populations reveals differences in expression of proteins regulating motility.

Lineage-marker depleted (Lin(-)) murine bone marrow cells expressing stem cell antigen 1 (Sca-1) were sorted on the basis of stem cell factor receptor (c-kit) expression to obtain Lin(-)Sca(+)Kit(+) or Lin(-)Sca(+)Kit(-) cells. Lin(-)Sca(+)Kit(-) cells have a markedly greater chemotactic response to stromal derived factor-1 (SDF-1). Using a novel fluorescent stain, we show that both populations generate similar levels of a key messenger, phosphatidylinositol 3,4,5 trisphosphate (PIP(3)), in response to SDF-1. Differences in motile behavior may therefore lie downstream of phosphatidylinositol 3-kinase (PI3-kinase) activation at the level of cytoskeleton regulation. The 2 cell populations were compared using 2-dimensional difference gel electrophoresis (2D-DIGE), with a maleimide CyDye fluorescent protein labeling technique that has enhanced sensitivity for low abundance samples. Comparative proteomic analysis of Cy3- and Cy5-labeled protein samples allows relative quantification of protein spots present in both cell populations; of these, 73% were common. Key protein differences were adseverin and gelsolin, actin micro-filament splicing proteins, regulated by Rac, downstream of PI3-kinase activation. Adseverin was shown to be acetylated, a novel modification for this protein. Differences in major regulators of cell shape and motility between the 2 populations can explain the differential response to SDF-1.

Adaptive morphological changes of neocortical interneurons in response to enlarged and more complex pyramidal cells in p21H-Ras(Val12) transgenic mice.

Morphological features of interneuronal adaptation to an altered, more complex neuronal architecture have been investigated in p21H-Ras(Val12) transgenic mice. This transgenic strain serves as a model for studying the morphogenetic role of the G-protein p21Ras on cortical principal neurons. We have recently demonstrated that postmitotic expression of constitutively active p21H-Ras(Val12) in the neocortical pyramidal cell population results in increased size and dendritic complexity of the affected neurons, leading to an enlarged cortical volume. Interneurons do not express the transgene and are therefore excluded from direct, intrinsic p21H-Ras(Val12) effects. In the present study, immunolabelling of gamma-amino-butyric-acid (GABA), and of the calcium-binding proteins parvalbumin, calbindin and calretinin revealed that in the transgenic mice local circuit neurons are not increased in either somal size or number and their main morphological characteristics are preserved. However, the dendritic arbour of interneurons was found to be extended, at least in the vertical dimension, to follow the cortical expansion. Immunostaining for the vesicular GABA transporter revealed a denser inhibitory innervation of p21H-Ras(Val12)-expressing pyramidal cell perikarya than in those of wild-type animals, while the overall density of inhibitory axon terminals within the cortex was decreased in the transgenic animals as a consequence of cortical expansion. The findings of the present study demonstrate the morphogenetic capacity of interneurons for adapting to morphological alterations of principal neurons in the cerebral cortex.

AHNAK interaction with the annexin 2/S100A10 complex regulates cell membrane cytoarchitecture.

Remodelling of the plasma membrane cytoarchitecture is crucial for the regulation of epithelial cell adhesion and permeability. In Madin-Darby canine kidney cells, the protein AHNAK relocates from the cytosol to the cytosolic surface of the plasma membrane during the formation of cell-cell contacts and the development of epithelial polarity. This targeting is reversible and regulated by Ca(2+)-dependent cell-cell adhesion. At the plasma membrane, AHNAK associates as a multimeric complex with actin and the annexin 2/S100A10 complex. The S100A10 subunit serves to mediate the interaction between annexin 2 and the COOH-terminal regulatory domain of AHNAK. Down-regulation of both annexin 2 and S100A10 using an annexin 2-specific small interfering RNA inhibits the association of AHNAK with plasma membrane. In Madin-Darby canine kidney cells, down-regulation of AHNAK using AHNAK-specific small interfering RNA prevents cortical actin cytoskeleton reorganization required to support cell height. We propose that the interaction of AHNAK with the annexin 2/S100A10 regulates cortical actin cytoskeleton organization and cell membrane cytoarchitecture.

Constitutive expression of p21H-Ras(Val12) in neurons induces increased axonal size and dendritic microtubule density in vivo.

The small G protein p21Ras is a key signal transducer mediating cellular growth and proliferation responses to extracellular stimuli. We investigated by electron microscopy the effects of augmented p21Ras activity on neuronal processes and microtubule arrangement in vivo. We used transgenic mice with a neuron-specific overexpression of p21H-RasVal12, which starts postnatally around Day 15. Axonal and dendritic diameters and the numerical density of dendritic microtubules were analyzed at postnatal Day 12 before the onset of transgene expression and in adult mice. In adult transgenic mice, calibers of both axons (corpus callosum) and dendrites (layers II/III of somatosensory cortex) were enlarged by about 57% and 79%, respectively. The increase in dendritic calibers was associated with an increment in the amount of microtubules. Even in dendrites of equivalent diameters, the number of microtubules was higher in transgenic mice compared to that in wild-type mice suggesting an elevated microtubule density. Changes in process diameters or microtubule density were not observed at postnatal Day 12 before relevant transcription of transgenic p21H-RasVal12. The present results extend previous findings on neuronal hypertrophy as a consequence of p21H-RasVal12 expression and suggest a profound influence on the dendritic microtubule network.

Unique tissue-specific level of DNA nucleotide excision repair in primary human mammary epithelial cultures.

DNA repair is essential for the maintenance of genomic integrity and stability. Nucleotide excision repair (NER) is a major pathway responsible for remediation of damage caused by UV light, bulky adducts, and cross-linking agents. We now show that NER capacity is differentially expressed in human tissues. We established primary cultures of peripheral blood lymphocytes (PBLs: N = 33) and foreskin fibroblasts (FF: N = 6), as well as adult breast tissue (N = 22) using a unique culture system, and measured their NER capacity using the unscheduled DNA synthesis (UDS) functional assay. Relative to FF, primary cultures of breast cells exhibited only 24.6 +/- 2.1% of NER capacity and PBLs only 8.9 +/- 1.2%. Cells from the breast therefore have a unique and distinctive DNA repair capacity. The NER capacities of all three cell types had similar coefficients of variation in the range of 10%-15%, which should be taken into account when running controls for this contextual assay. Unlike previous studies and speculation in the field, we found that NER was not affected by cell morphology, donor age, or proliferation as measured by the S phase index. While the NER capacity of the transformed lymphoblastoid cell line TK6 was within the range of our PBL samples, the breast tumor-derived MDA MB-231 cell line was four-fold higher than normal breast tissue. These studies show that analysis of baseline DNA repair in normal human cell types is critical as a basis for evaluation of the effects of "mutator" genes as etiological factors in the development of cancer.