Occupational lead exposure and survival with amyotrophic lateral sclerosis.

Objective: Lead exposure has been hypothesized to increase the risk of ALS, but only two studies have examined the association with ALS survival, and with inconsistent results. The use of occupational history to assess lead exposure can avoid reverse causation that may occur in epidemiologic analyses that use biomarkers of lead exposure collected after ALS onset.Methods: We evaluated the relationship of occupational lead exposure to ALS survival among 135 cases from an international ALS cohort that included deep phenotyping, careful follow-up, and questionnaires to quantify participants' occupation history. ALS patients were recruited in 2015-2019. We determined occupational lead exposure using a job-exposure matrix. We estimated hazard ratios (HR) and 95% confidence intervals (CI) for survival using Cox proportional hazard analysis with adjustment for covariates.Results: A total of 135 ALS patients completed the environmental questionnaires, among whom 38 reached a survival endpoint (death or permanent assisted ventilation). The median survival was 48.3 months (25th-75th percentile, 30.9-74.1). Older patients and those with initial symptom other than limb onset had shorter survival time. There were 36 ALS cases with occupational lead exposure. After adjusting for age, sex, site of onset, smoking, and military service, lead exposure was associated with an HR of 3.26 (95%CI 1.28-8.28). Results with adjustment for subsets of these covariates were similar.Conclusions: These results suggest that lead exposure prior to onset of ALS is associated with shorter survival following onset of ALS, and this association is independent of other prognostic factors.

Dynamic states of cervical epithelia during pregnancy and epithelial barrier disruption.

The cervical epithelium undergoes changes in proliferation, differentiation, and function that are critical to ensure fertility and maintain pregnancy. Here, we identify cervical epithelial subtypes in non-pregnant, pregnant, and in labor mice using single-cell transcriptome and spatial analysis. We identify heterogeneous subpopulations of epithelia displaying spatial and temporal specificity. Notably in pregnancy, two goblet cell subtypes are present in the most luminal layers with one goblet population expanding earlier in pregnancy than the other goblet population. The goblet populations express novel protective factors and distinct mucosal networks. Single-cell analysis in a model of cervical epithelial barrier disruption indicates untimely basal cell proliferation precedes the expansion of goblet cells with diminished mucosal integrity. These data demonstrate how the cervical epithelium undergoes continuous remodeling to maintain dynamic states of homeostasis in pregnancy and labor, and provide a framework to understand perturbations in epithelial health that increase the risk of premature birth.

Effect of infertility treatment and pregnancy-related hormones on breast cell proliferation in vitro.

BACKGROUND: Breast cancer development involves a series of mutations in a heterogeneous group of proto-oncogenes/tumor suppressor genes that alter mammary cells to create a microenvironment permissive to tumorigenesis. Exposure to hormones during infertility treatment may have a mutagenic effect on normal mammary epithelial cells, high-risk breast lesions and early-stage breast cancers. Our goal was to understand the association between infertility treatment and normal and cancerous breast cell proliferation. METHODS: MCF-10A normal mammary cells and the breast cancer cell lines MCF-7 [estrogen receptor (ER)-positive, well differentiated] and HCC 1937 (ER-negative, aggressive, BRCA1 mutation) were treated with the weak ER activator clomiphene citrate and hormones that are increased during infertility treatment. Direct effects of treatment on cell proliferation and colony growth were determined. RESULTS: While clomiphene citrate had no effect on MCF-10A cells or MCF-7 breast cancer cells, it decreased proliferation of HCC 1937 versus untreated cells (P= 0.003). Estrogen had no effect on either MCF-10A or HCC 1937 cells but, as expected, increased cell proliferation (20-100 nM; P≤0.002) and colony growth (10-30 nM; P< 0.0001) of MCF-7 cells versus control. Conversely, progesterone decreased both proliferation (P= 0.001) and colony growth (P= 0.01) of MCF-10A cells, inhibited colony size of MCF-7 cells (P= 0.01) and decreased proliferation of HCC 1937 cells (P= 0.008) versus control. hCG (100 mIU/ml) decreased both proliferation (P ≤ 0.01) and colony growth (P ≤ 0.002) of all three cell lines. CONCLUSIONS: Although these data are preclinical, they support possible indirect estrogenic effects of infertility regimens on ER-positive breast cancer cells and validate the potential protective effect of pregnancy-related exposure to hCG.

BpaB, a novel protein encoded by the Lyme disease spirochete's cp32 prophages, binds to erp Operator 2 DNA.

Borrelia burgdorferi produces Erp outer surface proteins throughout mammalian infection, but represses their synthesis during colonization of vector ticks. A DNA region 5' of the start of erp transcription, Operator 2, was previously shown to be essential for regulation of expression. We now report identification and characterization of a novel erp Operator 2-binding protein, which we named BpaB. erp operons are located on episomal cp32 prophages, and a single bacterium may contain as many as 10 different cp32s. Each cp32 family member encodes a unique BpaB protein, yet the three tested cp32-encoded BpaB alleles all bound to the same DNA sequence. A 20-bp region of erp Operator 2 was determined to be essential for BpaB binding, and initial protein binding to that site was required for binding of additional BpaB molecules. A 36-residue region near the BpaB carboxy terminus was found to be essential for high-affinity DNA-binding. BpaB competed for binding to erp Operator 2 with a second B. burgdorferi DNA-binding protein, EbfC. Thus, cellular levels of free BpaB and EbfC could potentially control erp transcription levels.

Revealing the Mutational Spectrum in Southern Africans With Amyotrophic Lateral Sclerosis.

BACKGROUND AND OBJECTIVES: To perform the first screen of 44 amyotrophic lateral sclerosis (ALS) genes in a cohort of African genetic ancestry individuals with ALS using whole-genome sequencing (WGS) data. METHODS: One hundred three consecutive cases with probable/definite ALS (using the revised El Escorial criteria), and self-categorized as African genetic ancestry, underwent WGS using various Illumina platforms. As population controls, 238 samples from various African WGS data sets were included. Our analysis was restricted to 44 ALS genes, which were curated for rare sequence variants and classified according to the American College of Medical Genetics guidelines as likely benign, uncertain significance, likely pathogenic, or pathogenic variants. RESULTS: Thirteen percent of 103 ALS cases harbored pathogenic variants; 5 different SOD1 variants (N87S, G94D, I114T, L145S, and L145F) in 5 individuals (5%, 1 familial case), pathogenic C9orf72 repeat expansions in 7 individuals (7%, 1 familial case) and a likely pathogenic ANXA11 (G38R) variant in 1 individual. Thirty individuals (29%) harbored ≥1 variant of uncertain significance; 10 of these variants had limited pathogenic evidence, although this was insufficient to permit confident classification as pathogenic. DISCUSSION: Our findings show that known ALS genes can be expected to identify a genetic cause of disease in >11% of sporadic ALS cases of African genetic ancestry. Similar to European cohorts, the 2 most frequent genes harboring pathogenic variants in this population group are C9orf72 and SOD1.

Borrelia burgdorferi EbfC defines a newly-identified, widespread family of bacterial DNA-binding proteins.

The Lyme disease spirochete, Borrelia burgdorferi, encodes a novel type of DNA-binding protein named EbfC. Orthologs of EbfC are encoded by a wide range of bacterial species, so characterization of the borrelial protein has implications that span the eubacterial kingdom. The present work defines the DNA sequence required for high-affinity binding by EbfC to be the 4 bp broken palindrome GTnAC, where 'n' can be any nucleotide. Two high-affinity EbfC-binding sites are located immediately 5' of B. burgdorferi erp transcriptional promoters, and binding of EbfC was found to alter the conformation of erp promoter DNA. Consensus EbfC-binding sites are abundantly distributed throughout the B. burgdorferi genome, occurring approximately once every 1 kb. These and other features of EbfC suggest that this small protein and its orthologs may represent a distinctive type of bacterial nucleoid-associated protein. EbfC was shown to bind DNA as a homodimer, and site-directed mutagenesis studies indicated that EbfC and its orthologs appear to bind DNA via a novel alpha-helical 'tweezer'-like structure.

Repeats expansions in ATXN2, NOP56, NIPA1 and ATXN1 are not associated with ALS in Africans.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized primarily by progressive loss of motor neurons. Although ALS occurs worldwide and the frequency and spectrum of identifiable genetic causes of disease varies across populations, very few studies have included African subjects. In addition to a hexanucleotide repeat expansion (RE) in C9orf72, the most common genetic cause of ALS in Europeans, REs in ATXN2, NIPA1 and ATXN1 have shown variable associations with ALS in Europeans. Intermediate range expansions in some of these genes (e.g. ATXN2) have been reported as potential risk factors, or phenotypic modifiers, of ALS. Pathogenic expansions in NOP56 cause spinocerebellar ataxia-36, which can present with prominent motor neuron degeneration. Here we compare REs in these genes in a cohort of Africans with ALS and population controls using whole genome sequencing data. Targeting genotyping of short tandem repeats at known loci within ATXN2, NIPA1, ATXN1 and NOP56 was performed using ExpansionHunter software in 105 Southern African (SA) patients with ALS. African population controls were from an in-house SA population control database (n = 25), the SA Human Genome Program (n = 24), the Simons Genome Diversity Project (n = 39) and the Illumina Polaris Diversity Cohort (IPDC) dataset (n = 50). We found intermediate RE alleles in ATXN2 (27-33 repeats) and ATXN1 (33-35 repeats), and NIPA1 long alleles (≥8 repeats) were rare in Africans, and not associated with ALS (p > 0.17). NOP56 showed no expanded alleles in either ALS or controls. We also compared the differences in allele distributions between the African and n = 50 European controls (from the IPDC). There was a statistical significant difference in the distribution of the REs in the ATXN1 between African and European controls (Chi-test p < 0.001), and NIPA1 showed proportionately more longer alleles (RE > 8) in Europeans vs. Africans (Fisher's p = 0.016). The distribution of RE alleles in ATXN2 and NOP56 were similar amongst African and European controls. In conclusion, repeat expansions in ATXN2, NIPA1 and ATXN1, which showed associations with ALS in Europeans, were not replicated in Southern Africans with ALS.

Borrelia burgdorferi RevA antigen binds host fibronectin.

Borrelia burgdorferi, the Lyme disease-causing spirochete, can persistently infect its vertebrate hosts for years. B. burgdorferi is often found associated with host connective tissue, where it interacts with components of the extracellular matrix, including fibronectin. Some years ago, a borrelial surface protein, named BBK32, was identified as a fibronectin-binding protein. However, B. burgdorferi BBK32 mutants are still able to bind fibronectin, indicating that the spirochete possesses additional mechanisms for adherence to fibronectin. We now demonstrate that RevA, an unrelated B. burgdorferi outer surface protein, binds mammalian fibronectin in a saturable manner. Site-directed mutagenesis studies identified the amino terminus of the RevA protein as being required for adhesion to fibronectin. RevA bound to the amino-terminal region of fibronectin. RevA binding to fibronectin was not inhibited by salt or heparin, suggesting that adhesin-ligand interactions are primarily nonionic and occur through the non-heparin-binding regions of the fibronectin amino-terminal domains. revA genes are widely distributed among Lyme disease spirochetes, and the present studies determined that all RevA alleles tested bound fibronectin. In addition, RevB, a paralogous protein found in a subset of B. burgdorferi strains, also bound fibronectin. We also confirmed that RevA is produced during mammalian infection but not during colonization of vector ticks and determined that revA transcription is controlled through a mechanism distinct from that of BBK32.

Borrelia burgdorferi infection-associated surface proteins ErpP, ErpA, and ErpC bind human plasminogen.

Host-derived plasmin plays a critical role in mammalian infection by Borrelia burgdorferi. The Lyme disease spirochete expresses several plasminogen-binding proteins. Bound plasminogen is converted to the serine protease plasmin and thereby may facilitate the bacterium's dissemination throughout the host by degrading extracellular matrix. In this work, we demonstrate plasminogen binding by three highly similar borrelial outer surface proteins, ErpP, ErpA, and ErpC, all of which are expressed during mammalian infection. Extensive characterization of ErpP demonstrated that this protein bound in a dose-dependent manner to lysine binding site I of plasminogen. Removal of three lysine residues from the carboxy terminus of ErpP significantly reduced binding of plasminogen, and the presence of a lysine analog, epsilon-aminocaproic acid, inhibited the ErpP-plasminogen interaction, thus strongly pointing to a primary role for lysine residues in plasminogen binding. Ionic interactions are not required in ErpP binding of plasminogen, as addition of excess NaCl or the polyanion heparin did not have any significant effect on binding. Plasminogen bound to ErpP could be converted to the active enzyme, plasmin. The three plasminogen-binding Erp proteins can also bind the host complement regulator factor H. Plasminogen and factor H bound simultaneously and did not compete for binding to ErpP, indicating separate binding sites for both host ligands and the ability of the borrelial surface proteins to bind both host proteins.

DNA-binding by Haemophilus influenzae and Escherichia coli YbaB, members of a widely-distributed bacterial protein family.

BACKGROUND: Genes orthologous to the ybaB loci of Escherichia coli and Haemophilus influenzae are widely distributed among eubacteria. Several years ago, the three-dimensional structures of the YbaB orthologs of both E. coli and H. influenzae were determined, revealing a novel "tweezer"-like structure. However, a function for YbaB had remained elusive, with an early study of the H. influenzae ortholog failing to detect DNA-binding activity. Our group recently determined that the Borrelia burgdorferi YbaB ortholog, EbfC, is a DNA-binding protein. To reconcile those results, we assessed the abilities of both the H. influenzae and E. coli YbaB proteins to bind DNA to which B. burgdorferi EbfC can bind. RESULTS: Both the H. influenzae and the E. coli YbaB proteins bound to tested DNAs. DNA-binding was not well competed with poly-dI-dC, indicating some sequence preferences for those two proteins. Analyses of binding characteristics determined that both YbaB orthologs bind as homodimers. Different DNA sequence preferences were observed between H. influenzae YbaB, E. coli YbaB and B. burgdorferi EbfC, consistent with amino acid differences in the putative DNA-binding domains of these proteins. CONCLUSION: Three distinct members of the YbaB/EbfC bacterial protein family have now been demonstrated to bind DNA. Members of this protein family are encoded by a broad range of bacteria, including many pathogenic species, and results of our studies suggest that all such proteins have DNA-binding activities. The functions of YbaB/EbfC family members in each bacterial species are as-yet unknown, but given the ubiquity of these DNA-binding proteins among Eubacteria, further investigations are warranted.

Borrelia burgdorferi complement regulator-acquiring surface proteins (BbCRASPs): Expression patterns during the mammal-tick infection cycle.

Host complement is widely distributed throughout mammalian body fluids and can be activated immediately as part of the first line of defense against invading pathogens. The agent of Lyme disease, Borrelia burgdorferi sensu lato (s.l.), is naturally resistant to that innate immune defense system of its hosts. One resistance mechanism appears to involve binding fluid-phase regulators of complement to distinct borrelial outer surface molecules known as CRASPs (complement regulator acquiring surface proteins). Using sensitive molecular biology techniques, expression patterns of all three classes of genes encoding the CRASPs of B. burgdorferi sensu stricto (BbCRASPs) have been analyzed throughout the natural tick-mammal infection cycle. Each class shows a different expression profile in vivo and the results are summarized herein. Studies on the expression of B. burgdorferi genes using animal models of infection have advanced our knowledge on the ability of the causative agent to circumvent innate immune defenses, the contributions of CRASPs to spirochete infectivity, and the pathogenesis of Lyme disease.

Lyme borreliosis spirochete Erp proteins, their known host ligands, and potential roles in mammalian infection.

Lyme borreliae naturally maintain numerous distinct DNA elements of the cp32 family, each of which carries a mono- or bicistronic erp locus. The encoded Erp proteins are surface-exposed outer membrane lipoproteins that are produced at high levels during mammalian infection but largely repressed during colonization of vector ticks. Recent studies have revealed that some Erp proteins can serve as bacterial adhesins, binding host proteins such as the complement regulator factor H and the extracellular matrix component laminin. These results suggest that Erp proteins play roles in multiple aspects of mammalian infection.

Production of outer surface protein A by Borrelia burgdorferi during transmission from infected mammals to feeding ticks is insufficient to trigger OspA seroconversion.

The Lyme disease spirochete, Borrelia burgdorferi, produces two outer surface lipoproteins, OspA and OspB, that are essential for colonization of tick vectors. Both proteins are highly expressed during transmission from infected mammals to feeding ticks and during colonization of tick midguts, but are repressed when bacteria are transmitted from ticks to mammals. Humans and other infected mammals generally do not produce antibodies against either protein, although some Lyme disease patients do seroconvert and produce antibodies against OspA for unknown reasons. We hypothesized that, if such patients had been fed upon by additional ticks, bacteria moving from the patients' bodies to the feeding ticks would have produced OspA and OspB proteins, which then led to immune system recognition and antibody production. This hypothesis was tested by analyzing immune responses of infected mice following feedings by additional Ixodes scapularis ticks. However, results of the present studies demonstrate that expression of OspA and OspB by B. burgdorferi during transmission from infected mammals to feeding ticks does not trigger seroconversion.

Frequent sgRNA-barcode recombination in single-cell perturbation assays.

Simultaneously detecting CRISPR-based perturbations and induced transcriptional changes in the same cell is a powerful approach to unraveling genome function. Several lentiviral approaches have been developed, some of which rely on the detection of distally located genetic barcodes as an indirect proxy of sgRNA identity. Since barcodes are often several kilobases from their corresponding sgRNAs, viral recombination-mediated swapping of barcodes and sgRNAs is feasible. Using a self-circularization-based sgRNA-barcode library preparation protocol, we estimate the recombination rate to be ~50% and we trace this phenomenon to the pooled viral packaging step. Recombination is random, and decreases the signal-to-noise ratio of the assay. Our results suggest that alternative approaches can increase the throughput and sensitivity of single-cell perturbation assays.

Identification of compound heterozygous variants in OPTN in an ALS-FTD patient from the CReATe consortium: a case report.

Homozygous loss-of-function mutations in optineurin (OPTN) are a rare cause of amyotrophic lateral sclerosis (ALS), whereas heterozygous loss-of-function mutations have been suggested to increase ALS disease risk. We report a patient with ALS and frontotemporal dementia (FTD) from the Clinical Research in ALS and Related Disorders for Therapeutic Development (CReATe) Consortium carrying compound heterozygous loss-of-function variants in OPTN. Quantitative real-time mRNA expression analyses revealed a 75-80% reduction in OPTN expression in blood in the OPTN carrier as compared to controls, suggesting at least partial nonsense-mediated decay of the mutant transcripts. This case report illustrates the diverse inheritance patterns and variable clinical presentations associated with OPTN mutations, and underscores the importance of complete OPTN gene screening in patients with ALS and related disorders, especially in the context of clinical genetic testing.

Evolving models of Lyme disease spirochete gene regulation.

The spirochete Borrelia burgdorferi, the causative agent of Lyme disease (Lyme borreliosis), is well-adapted to maintain a natural cycle of alternately infecting vertebrates and blood-sucking ticks. During this cycle, B. burgdorferi interacts with a broad spectrum of vertebrate and arthropod tissues, acquires nutrients in diverse environments and evades killing by vertebrate and tick immune systems. The bacterium also senses when situations occur that necessitate transmission between hosts, such as when an infected tick is taking a blood meal from a potential host. To accurately accomplish the requirements necessary for survival in nature, B. burgdorferi must be keenly aware of its surroundings and respond accordingly. In this review, we trace studies performed to elucidate regulatory mechanisms employed by B. burgdorferi to control gene expression, and the development of models or "paradigms" to explain experimental results. Through comparisons of five borrelial gene families, it is readily apparent that each is controlled through a distinct mechanism. Furthermore, those results indicate that current models of interpreting in vitro data cannot accurately predict all aspects of B. burgdorferi environmental sensing and gene regulation in vivo.

Presymptomatic ALS genetic counseling and testing: Experience and recommendations.

Remarkable advances in our understanding of the genetic contributions to amyotrophic lateral sclerosis (ALS) have sparked discussion and debate about whether clinical genetic testing should routinely be offered to patients with ALS. A related, but distinct, question is whether presymptomatic genetic testing should be offered to family members who may be at risk for developing ALS. Existing guidelines for presymptomatic counseling and testing are mostly based on small number of individuals, clinical judgment, and experience from other neurodegenerative disorders. Over the course of the last 8 years, we have provided testing and 317 genetic counseling sessions (including predecision, pretest, posttest, and ad hoc counseling) to 161 first-degree family members participating in the Pre-Symptomatic Familial ALS Study (Pre-fALS), as well as testing and 75 posttest counseling sessions to 63 individuals with familial ALS. Based on this experience, and the real-world challenges we have had to overcome in the process, we recommend an updated set of guidelines for providing presymptomatic genetic counseling and testing to people at high genetic risk for developing ALS. These recommendations are especially timely and relevant given the growing interest in studying presymptomatic ALS.

Impact of infertility regimens on breast cancer cells: follicle-stimulating hormone and luteinizing hormone lack a direct effect on breast cell proliferation in vitro.

OBJECTIVE: To examine the impact of hormones used for controlled ovarian hyperstimulation (COH) on normal and malignant breast cell growth and proliferation. DESIGN: In vitro study of cultured normal and malignant breast cell lines. SETTING: Academic medical center. PATIENT(S): None. INTERVENTION(S): Normal and malignant breast cell lines cultured in two- and three-dimensional (2D and 3D) systems and treated with follicle-stimulating hormone (FSH), luteinizing hormone (LH), or FSH with LH or human chorionic gonadotropin (hCG). MAIN OUTCOME MEASURE(S): Effects of treatment on cell proliferation in 2D culture using the MTS assay and on colony growth in 3D culture. RESULT(S): Compared with untreated cells, normal MCF-10A cells showed a decrease in proliferation and colony size when exposed to a combination of FSH and hCG. The HCC 1937 cells treated with FSH and LH also showed a decrease in colony growth but no change in proliferation. None of the treatments had an effect on the proliferation or colony size of the MCF-7 cells. CONCLUSION(S): Follicle-stimulating hormone, LH, and hCG do not appear to cause an increase in cell proliferation or colony growth in either normal or malignant mammary epithelial cell lines. The potential risk for mammary cell transformation associated with these agents may be related to indirect endocrine effects on breast cell physiology.

Impact of cyclin E overexpression on Smad3 activity in breast cancer cell lines.

Smad3, a component of the TGFß signaling pathway, contributes to G1 arrest in breast cancer cells. Overexpression of the cell cycle mitogen, cyclin E, is associated with poor prognosis in breast cancer, and cyclin E/CDK2 mediated phosphorylation of Smad3 has been linked with inhibition of Smad3 activity. We hypothesized that the biological aggressiveness of cyclin E overexpressing breast cancer cells would be associated with CDK2 phosphorylation and inhibition of the tumor suppressant action of Smad3. Expression constructs containing empty vector, wild type (WT) Smad3, or Smad3 with CDK phosphorylation site mutations were co-transfected with a Smad3-responsive reporter construct into parental, vector control (A1), or cyclin E overexpressing (EL1) MCF7 cells. Smad3 function was evaluated by luciferase reporter assay and mRNA analysis. The impact of a Cdk2 inhibitor and cdk2 siRNA on Smad3 activity was also assessed. Cells expressing Smad3 containing mutations of the CDK phosphorylation sites had higher p15 and p21 and lower c-myc mRNA levels, as well as higher Smad3-responsive reporter activity, compared with controls or cells expressing WT Smad3. Transfection of cdk2 siRNA resulted in a significant increase in Smad3-responsive reporter activity compared with control siRNA; reporter activity was also increased after the treatment with a Cdk2 inhibitor. Thus, cyclin E-mediated inhibition of Smad3 is regulated by CDK2 phosphorylation of the Smad3 protein in MCF7 cells. Inhibition of CDK2 may lead to restoration of Smad3 tumor suppressor activity in breast cancer cells, and may represent a potential treatment approach for cyclin E overexpressing breast cancers.

Cyclin-dependent kinase 4-mediated phosphorylation inhibits Smad3 activity in cyclin D-overexpressing breast cancer cells.

Smad3, a component of the transforming growth factor ß signaling cascade, contributes to G(1) arrest in breast cancer cells. Cyclin D1/cyclin-dependent kinase 4 (CDK4) promotes G(1)-S-phase transition, and CDK phosphorylation of Smad3 has been associated with inhibition of Smad3 activity. We hypothesized that overexpression of cyclin D1 exerts tumorigenic effects in breast cancer cells through CDK4-mediated phosphorylation and inhibition of Smad3 and release of G(1) arrest. Real-time quantitative reverse transcription-PCR and immunoblotting were used to evaluate expression of study proteins in cyclin D1-overexpressing breast cancer cells. Smad3 transcriptional activity and cell cycle control were examined in cells transfected with wild-type (WT) Smad3 or Smad3 with single or multiple CDK phosphorylation site mutations (M) in the presence or absence of the CDK4 inhibitor or cotransfection with cdk4 small interfering RNA (siRNA). Transfection of the Smad3 5M construct resulted in decreased c-myc and higher p15(INK4B) expression. Compared with WT Smad3, overexpression of the Smad3 T8, T178, 4M, or 5M mutant constructs resulted in higher Smad3 transcriptional activity. Compared with cells transfected with WT Smad3, Smad3 transcriptional activity was higher in cells overexpressing Smad3 mutant constructs and treated with the CDK4 inhibitor or transfected with cdk4 siRNA. Cells transfected with Smad3 T8 or T178 and treated with the CDK4 inhibitor showed an increase in the G(1) cell population. Inhibition of CDK-mediated Smad3 phosphorylation released cyclin D1-regulated blockade of Smad3 transcriptional activity and recovered cell cycle arrest in breast cancer cells. Targeted inhibition of CDK4 activity may have a role in the treatment of cyclin D-overexpressing breast cancers.