NuMA Expression Research Articles

Kinesin KIF3A regulates meiotic progression and spindle assembly in oocyte meiosis

Kinesin family member 3A (KIF3A) is a microtubule-oriented motor protein that belongs to the kinesin-2 family for regulating intracellular transport and microtubule movement. In this study, we characterized the critical roles of KIF3A during mouse oocyte meiosis. We found that KIF3A associated with microtubules during meiosis and depletion of KIF3A resulted in oocyte maturation defects. LC–MS data indicated that KIF3A associated with cell cycle regulation, cytoskeleton, mitochondrial function and intracellular transport-related molecules. Depletion of KIF3A activated the spindle assembly checkpoint, leading to metaphase I arrest of the first meiosis. In addition, KIF3A depletion caused aberrant spindle pole organization based on its association with KIFC1 to regulate expression and polar localization of NuMA and γ-tubulin; and KIF3A knockdown also reduced microtubule stability due to the altered microtubule deacetylation by histone deacetylase 6 (HDAC6). Exogenous Kif3a mRNA supplementation rescued the maturation defects caused by KIF3A depletion. Moreover, KIF3A was also essential for the distribution and function of mitochondria, Golgi apparatus and endoplasmic reticulum in oocytes. Conditional knockout of epithelial splicing regulatory protein 1 (ESRP1) disrupted the expression and localization of KIF3A in oocytes. Overall, our results suggest that KIF3A regulates cell cycle progression, spindle assembly and organelle distribution during mouse oocyte meiosis.

Investigation of the SPAG5 gene expression and amplification related to the NuMA mRNA levels in breast ductal carcinoma

BackgroundThe cell proliferative markers are very important in breast cancer. Since SPAG5 and NuMA proteins play a significant role in the mitosis regulatory network and cell division, we aimed to study their mRNA levels as well as SPAG5 gene amplification correlated to clinicopathological status in ductal carcinoma of the breast.MethodsSPAG5 and NuMA gene expressions were investigated in 40 breast cancer tissues and normal adjacent tissues via real-time PCR. PUM1 was selected as the reference gene. QMF PCR method was applied to study SPAG5 gene amplification and AGBL2, BOD1L, and POR were designated as internal control genes. Gene amplification was determined by calculating a dosage quotient for each DNA fragment.ResultsIncreased SPAG5 mRNA expression was detected in breast cancer tissues (p = 0.005) and related to tumor size. No significant difference was observed between NuMA gene expression level in tumor tissue and the normal adjacent tissue (p = 0.56). However, we observed that NuMA expression was significantly increased in ER-positive tumor tissues. There was no clear correlation pattern between SPAG5 and NuMA mRNA levels (r = 0.33). Seventeen percent of tissues showed complete amplification in SPAG5 gene fragments.ConclusionOur results were consistent with the previous publications regarding SPAG5 gene expression and amplification in breast cancer with an emphasis on the prominent role of this protein in tumor pathogenesis. Our results failed to yield any correlation between SPAG5 and NuMA mRNA levels which implies independence of these genes in breast cancer pathogenesis.

The nuclear structural protein NuMA is a negative regulator of 53BP1 in DNA double-strand break repair.

P53-binding protein 1 (53BP1) mediates DNA repair pathway choice and promotes checkpoint activation. Chromatin marks induced by DNA double-strand breaks and recognized by 53BP1 enable focal accumulation of this multifunctional repair factor at damaged chromatin. Here, we unveil an additional level of regulation of 53BP1 outside repair foci. 53BP1 movements are constrained throughout the nucleoplasm and increase in response to DNA damage. 53BP1 interacts with the structural protein NuMA, which controls 53BP1 diffusion. This interaction, and colocalization between the two proteins in vitro and in breast tissues, is reduced after DNA damage. In cell lines and breast carcinoma NuMA prevents 53BP1 accumulation at DNA breaks, and high NuMA expression predicts better patient outcomes. Manipulating NuMA expression alters PARP inhibitor sensitivity of BRCA1-null cells, end-joining activity, and immunoglobulin class switching that rely on 53BP1. We propose a mechanism involving the sequestration of 53BP1 by NuMA in the absence of DNA damage. Such a mechanism may have evolved to disable repair functions and may be a decisive factor for tumor responses to genotoxic treatments.

Abstract 3789: Genomic instability changes acquired by HPV16 E6/E7 targeting multipolar mitoses aneuploidy: Cellular sequelae

Abstract Malignant progression of high-risk Papillomavirus (HPV), associated lesions is a slow and inefficient process. HPV a non-enveloped epitheliotropic double stranded DNA virus, is an etiological agent of oral cancer. Oral cancer is related to the persistent infection by high-risk HPV type 16, E6/E7 oncoproteins. The E6/E7 complex significantly contribute to the carcinogenic genetic instability effect of high-risk HPV through the degradation of two gatekeeper proteins, p53 and pRB, respectively. This process is associated with viral genome integration into a host cellular chromosome and accumulation of numerical and structural chromosome aberrations. Viral genome integration results in consistent, sustained and dysregulated expression of the E6 and E7 oncogenes, and continued E6/E7 expression is necessary for maintenance of the transformed phenotype of HPV positive cancer cell. Induction of genomic instability by high-risk HPV E6/E7 demonstrates that HPV also drive malignant progression through targeting the nuclear and mitotic apparatus protein-1 (NuMA), and through subversion of DNA double strand break repair by HPV E6/E7 induced centrosome abnormalities. This in turn may cause abnormal localization of the microtubule protein dynein. The NuMA provides a cohesive force that guarantees the integrity of the mitotic spindle poles. However, there are no studies addressing the relationship between HPV16 infection-oral cancer, NuMA expression, and genomic instability. The aim of this study determined if HPV16 E6/E7 subverts centrosome coalescence in oral cancer caused by HPV oncogenes targeting the NuMA protein. In our experimental procedures, we proposed a novel approach to study HPV-induced chromosomal changes with reconstructed human oral epithelium in SCID mouse. We used HPV16, which is the most common HPV type found in oral cancer. Two plasmids were used to produce HPV-16 E6/E7. The first plasmid p16sheLL expresses the two viral capsid proteins, L1 and L2. The second plasmid pBR322HPV16 contains the full length HPV-16 genome. This approach closely mimics the architecture of normal human oral epithelium. In our study, we first focused on analyzing selected cellular processes involving multipolar mitoses. We examined Dynein localization in mitotic cells by immunofluorescence. We also analyzed progressive morphological and cytopathic changes in HPV-infected oral epithelia by using molecular cytogenetic analysis. In summary these data indicate that NuMA expression is alternated, and HPV16 E6/E7 oncoproteins can promote increased upregulation of genomic instability by induction of centrosome abnormalities and inhibition of DNA double strand break repair. We also found High-risk HPV16 E6/E7 oncoproteins induced mitotic defects and genetic aneuploidies through induction of supernumerary anaphase bridges, supernumerary centrosomes, and DNA double strand breaks. Citation Format: Eva McGhee, Mengtao Li, Yi-Ling Lin, Khadijah Lang, Meidrah Tyler, Judith Okoro, Mai Do, Naomi Long, Jenna Cormier, Adin Handler, Julian Handler, Sameeran Das, Benjamin Liu, Anthonia Duru, Billy Ballard, Roland Pattillo, Jay Vadgama. Genomic instability changes acquired by HPV16 E6/E7 targeting multipolar mitoses aneuploidy: Cellular sequelae [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3789.

The nuclear structural protein NuMA is a negative regulator of 53BP1 in DNA double-strand break repair.

P53-binding protein 1 (53BP1) mediates DNA repair pathway choice and promotes checkpoint activation. Chromatin marks induced by DNA double-strand breaks and recognized by 53BP1 enable focal accumulation of this multifunctional repair factor at damaged chromatin. Here, we unveil an additional level of regulation of 53BP1 outside repair foci. 53BP1 movements are constrained throughout the nucleoplasm and increase in response to DNA damage. 53BP1 interacts with the structural protein NuMA, which controls 53BP1 diffusion. This interaction, and colocalization between the two proteins in vitro and in breast tissues, is reduced after DNA damage. In cell lines and breast carcinoma NuMA prevents 53BP1 accumulation at DNA breaks, and high NuMA expression predicts better patient outcomes. Manipulating NuMA expression alters PARP inhibitor sensitivity of BRCA1-null cells, end-joining activity, and immunoglobulin class switching that rely on 53BP1. We propose a mechanism involving the sequestration of 53BP1 by NuMA in the absence of DNA damage. Such a mechanism may have evolved to disable repair functions and may be a decisive factor for tumor responses to genotoxic treatments.

The nuclear mitotic apparatus protein NuMA controls rDNA transcription and mediates the nucleolar stress response in a p53-independent manner.

The nuclear mitotic apparatus protein, NuMA, is involved in major cellular events such as DNA damage response, apoptosis and p53-mediated growth-arrest, all of which are under the control of the nucleolus upon stress. Proteomic investigation has identified NuMA among hundreds of nucleolar proteins. Yet, the precise link between NuMA and nucleolar function remains undetermined. We confirm that NuMA is present in the nucleolus and reveal redistribution of NuMA upon actinomycin D or doxorubicin-induced nucleolar stress. NuMA coimmunoprecipitates with RNA polymerase I, with ribosomal proteins RPL26 and RPL24, and with components of B-WICH, an ATP-dependent chromatin remodeling complex associated with rDNA transcription. NuMA also binds to 18S and 28S rRNAs and localizes to rDNA promoter regions. Downregulation of NuMA expression triggers nucleolar stress, as shown by decreased nascent pre-rRNA synthesis, fibrillarin perinucleolar cap formation and upregulation of p27kip1, but not p53. Physiologically relevant nucleolar stress induction with reactive oxygen species reaffirms a p53-independent p27kip1 response pathway and leads to nascent pre-rRNA reduction. It also promotes the decrease in the amount of NuMA. This previously uncharacterized function of NuMA in rDNA transcription and p53-independent nucleolar stress response supports a central role for this nuclear structural protein in cellular homeostasis.

The Short Isoform of Nuclear Mitotic Apparatus Protein 1 Functions as a Putative Tumor Suppressor.

Background:Nuclear mitotic apparatus protein 1 (NuMA1) had been reported to produce three groups of isoforms categorized as long, middle, and short groups, of which short NuMA displayed distinct localization patterns compared to long and middle isoforms. However, the function of short NuMA was not clear in the progress of cancer formation. This study aimed to unveil the role of short NuMA in cancer pathogenesis.Methods:The expression levels of short isoforms were explored in paired gastric carcinoma (GC) samples and different cell lines. Furthermore, the short isoform behaved as a putative tumor suppressor based on cell proliferation and cell colony formation assays. Pull-down assay and whole-genome gene expression analysis were carried out to search candidate interaction partners of short NuMA.Results:The expression of short NuMA was highly expressed in S and G2 phases of the cell cycle; compared with nontumor tissues, short NuMA downregulated in nine GCs (GC1 [0.131, P = 5 × 10−4]; GC2 [0.316, P = 3 × 10−5]; GC3 [0.111, P = 6 × 10−4]; GC4 [0.456, P = 0.011]; GC5 [0.474, P = 0.001]; GC6 [0.311, P = 0.004]; GC7 [0.28, P = 3 × 10−5]; GC8 [0.298, P = 0.007]; and GC9 [0.344, P = 0.002]). Besides, high expression of short NuMA significantly inhibits cell growth (2.43 × 105 vs. 2.97 × 105, P = 0.0029) and cell clone information in vitro (70 vs. 2, P = 1.67 × 10−45). Short NuMA could bind with alpha–actinin-4 (ACTN4), a putative tumor promoting gene. Overexpression of short NuMA could tremendously decrease the expression of MYB proto-oncogene like 2 (MYBL2) of about 92-fold, which played an important role in the cell cycles.Conclusions:Short isoform of NuMA might be functioned as a putative role of tumor suppressor. Further studies should be made to illuminate the relationship between ACTN4, MYBL2, and tumor progression.

Abstract 2755: Regulation of 53BP1 by the structural nuclear protein NuMA

Abstract DNA double-strand breaks (DSBs) can cause genomic instability and cancer through point mutations and genomic translocations if they are not properly repaired. DSBs are repaired by two major pathways: homologous recombination (HR) or nonhomologous end joining (NHEJ). The cellular choice between these two pathways is critical to cell survival and is altered in cancers. 53BP1, which promotes NHEJ by blocking CtIP-dependent DNA resection, is a key protein mediating repair pathway choice. Here, we present a new negative regulation mechanism of 53BP1 that relies on the structural nuclear protein NuMA. Apart from its role in mitotic spindle assembly, NuMA influences chromatin organization during the interphase and modulates the chromatin response to DNA damage. We identified an interaction between NuMA and 53BP1 by proteomics analysis of a 53BP1 pull-down and confirmed this interaction by NuMA immunoprecipitation. We measured greater than 50% decreased 53BP1-NuMA binding in response to DSB, which suggest that, in the absence of DNA damage, NuMA may restrain 53BP1 diffusion. This hypothesis is supported by fluorescence correlation spectroscopy (FCS) measurements in cells expressing GFP-tagged 53BP1, as well as by microirradiation experiments in which NuMA overexpression or silencing inhibited or promoted 53BP1 recruitment at DNA damage sites, respectively. 53BP1 has an essential role in B cell class switch recombination (CSR) and mediates PARPi sensitivity in BRCA1-null cells. We will present how modulating NuMA expression affects both aspects of 53BP1 function. The results from our work will help to understand how 53BP1 is controlled, and shed light on the mechanisms regulating this modulator of PARPi sensitivity in the presence or absence of DNA damage. Citation Format: Naike Salvador Moreno. Regulation of 53BP1 by the structural nuclear protein NuMA. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2755.

NuMA Overexpression in Epithelial Ovarian Cancer

Highly aneuploid tumours are common in epithelial ovarian cancers (EOC). We investigated whether NuMA expression was associated with this phenomenon.NuMA protein levels in normal and tumour tissues, ovarian cell lines and primary cultures of malignant cells derived from ovarian ascitic fluids were analysed by Affymetrix microarray analysis, immunoblotting, immunohistochemistry (IHC) and immunofluorescence (IF), with results correlated to associated clinical data. Aneuploidy status in primary cultures was determined by FACS analysis.Affymetrix microarray data indicated that NuMA was overexpressed in tumour tissue, primary cultures and cell lines compared to normal ovarian tissue. IHC revealed low to weak NuMA expression in normal tissues. Expression was upregulated in tumours, with a significant association with disease stage in mucinous EOC subtypes (p = 0.009), lymph node involvement (p = 0.03) and patient age (p = 0.04). Additional discontinuous data analysis revealed that high NuMA levels in tumours decreased with grade (p = 0.02) but increased with disease stage (p = 0.04) in serous EOC. NuMA expression decreased in late disease stage 4 endometrioid EOCs. High NuMA levels decreased with increased tumour invasion in all subtypes (p = 0.03). IF of primary cultures revealed that high NuMA levels at mitotic spindle poles were significantly associated with a decreased proportion of cells in cytokinesis (p = 0.05), increased binucleation (p = 0.021) and multinucleation (p = 0.007), and aneuploidy (p = 0.008).NuMA is highly expressed in EOC tumours and high NuMA levels correlate with increases in mitotic defects and aneuploidy in primary cultures.

GAS41 amplification results in overexpression of a new spindle pole protein

Amplification is a hallmark of many human tumors but the role of most amplified genes in human tumor development is not yet understood. Previously, we identified a frequently amplified gene in glioma termed glioma-amplified sequence 41 (GAS41). Using the TCGA data portal and performing experiments on HeLa and TX3868, we analyzed the role of GAS41 amplification on GAS41 overexpression and the effect on the cell cycle. Here we show that GAS41 amplification is associated with overexpression in the majority of cases. Both induced and endogenous overexpression of GAS41 leads to an increase in multipolar spindles. We showed that GAS41 is specifically associated with pericentrosome material. As result of an increased GAS41 expression we found bipolar spindles with misaligned chromosomes. This number was even increased by a combined overexpression of GAS41 and a reduced expression of NuMA. We propose that GAS41 amplification may have an effect on the highly altered karyotype of glioblastoma via its role during spindle pole formation.

Kaposi's Sarcoma-Associated Herpesvirus-Encoded LANA Can Interact with the Nuclear Mitotic Apparatus Protein To Regulate Genome Maintenance and Segregation

Kaposi's sarcoma-associated herpesvirus (KSHV) genomes are tethered to the host chromosomes and partitioned faithfully into daughter cells with the host chromosomes. The latency-associated nuclear antigen (LANA) is important for segregation of the newly synthesized viral genomes to the daughter nuclei. Here, we report that the nuclear mitotic apparatus protein (NuMA) and LANA can associate in KSHV-infected cells. In synchronized cells, NuMA and LANA are colocalized in interphase cells and separate during mitosis at the beginning of prophase, reassociating again at the end of telophase and cytokinesis. Silencing of NuMA expression by small interfering RNA and expression of LGN and a dominant-negative of dynactin (P150-CC1), which disrupts the association of NuMA with microtubules, resulted in the loss of KSHV terminal-repeat plasmids containing the major latent origin. Thus, NuMA is required for persistence of the KSHV episomes in daughter cells. This interaction between NuMA and LANA is critical for segregation and maintenance of the KSHV episomes through a temporally controlled mechanism of binding and release during specific phases of mitosis.

Dictyostelium nucleomorphin is a member of the BRCT-domain family of cell cycle checkpoint proteins

A search of the Dictyostelium genome project database (http://dictybase.org/db/cgi-bin/blast.pl) with nucleomorphin, a protein that regulates the nuclear number, predicted it to be encoded by a larger gene containing a putative breast cancer carboxy-terminus domain (BRCT). Using RT-PCR, Northern and Western blotting we have identified a differentially expressed, 2318 bp cDNA encoding a protein isoform of Dictyostelium NumA with an apparent molecular weight of 70 kDa that we have called NumB. It contains a single amino-terminal BRCT-domain spanning residues 125-201. Starvation of shaking cultures reduces NumA expression by approximately 88+/-5.6%, whereas NumB expression increases approximately 35+/-3.5% from vegetative levels. NumC, a third isoform that is also expressed during development but not growth, remains to be characterized. These findings suggest NumB may be a member of the BRCT-domain containing cell cycle checkpoint proteins.

NuMA in rat testis—Evidence for roles in proliferative activity and meiotic cell division

NuMA is a well-characterized organizer of the mitotic spindle, which is believed to play a structural role in interphase nucleus. We studied the expression of NuMA in rat seminiferous epithelium in detail. Different stages of the cycle of the seminiferous epithelium were identified using transillumination. Corresponding areas were microdissected and analysed using immunofluorescence, immunohistochemistry, or immunoblotting. NuMA was expressed in Sertoli cells, proliferating type A and B spermatogonia, and early spermatids but it was absent in late spermatids and mature spermatozoa. Interestingly, NuMA-positive primary spermatocytes lost their nuclear NuMA at the beginning of long-lasting prophase of the first meiotic division. A strong expression was again observed at the end of the prophase and finally, a redistribution of NuMA into pole regions of the meiotic spindle was observed in first and second meiotic divisions. In immunoblotting, a single 250-kDa protein present in all stages of the rat seminiferous epithelial cycle was detected. Our results show that NuMA is not essential for the organization of nuclear structure in all cell types and suggest that its presence is more likely connected to the proliferation phase of the cells. They also suggest that NuMA may play an important role in meiotic cell division.

NuMA expression and function in mouse oocytes and early embryos.

Nuclear mitotic apparatus protein (NuMA), originally described as a nuclear protein, is an essential component in the formation and maintenance of mitotic spindle poles. In this study, we analyze the expression pattern and function of NuMA in mouse oocytes and early embryos. In germinal vesicle-stage oocytes, NuMA was detected both at the centrosome and in the nucleus. However, after nuclear maturation and extrusion of the first polar body, NuMA was concentrated at the broad meiotic spindle poles and at cytasters (centers of cytoplasmic microtubule asters) of mature metaphase II oocytes. Cold-induced depolymerization of microtubules appeared to disassociate NuMA foci from the cytoplasmic cytasters. During fertilization, NuMA was relocated into the re-formed male and female pronuclei. Microinjection of anti-NuMA antibody into 1 of 2 cells of 2-cell-stage embryos inhibited normal cell division. These results suggest that NuMA might play an important role in cell division during early embryonic mitosis.

NuMA Expression and Function in Mouse Oocytes and Early Embryos

Nuclear mitotic apparatus protein (NuMA), originally described as a nuclear protein, is an essential component in the formation and maintenance of mitotic spindle poles. In this study, we analyze the expression pattern and function of NuMA in mouse oocytes and early embryos. In germinal vesicle-stage oocytes, NuMA was detected both at the centrosome and in the nucleus. However, after nuclear maturation and extrusion of the first polar body, NuMA was concentrated at the broad meiotic spindle poles and at cytasters (centers of cytoplasmic microtubule asters) of mature metaphase II oocytes. Cold-induced depolymerization of microtubules appeared to disassociate NuMA foci from the cytoplasmic cytasters. During fertilization, NuMA was relocated into the re-formed male and female pronuclei. Microinjection of anti-NuMA antibody into 1 of 2 cells of 2-cell-stage embryos inhibited normal cell division. These results suggest that NuMA might play an important role in cell division during early embryonic mitosis.

Signaling through Cdk2, importin-α and NuMA is required for H 2O 2-induced mitosis in primary type II pneumocytes

Proliferation of alveolar type II pneumocytes, the multipotent stem cells of the alveoli, has been implicated in the development of lung adenocarcinoma. Hydrogen peroxide (H 2O 2), a potent promoter of signaling cascades, can mediate the transmission of many intracellular signals including those involved in cell proliferation. In this study using rat primary type II pneumocytes, we demonstrate that H 2O 2 significantly increases mitosis through a pathway that includes cyclin-dependent kinase 2 (Cdk2); importin-α, a nuclear trafficking regulator; and nuclear mitotic apparatus protein (NuMA), an essential component in mitotic spindle pole formation. Upon H 2O 2 treatment, Cdk2 is phosphorylated at position thr-160 leading to increases in importin-α and NuMA protein levels and resulting in a significant increase of G 2/M phase in a roscovatine-dependent manner. Type II pneumocytes transfected with NuMA cDNA also show significant increases in G 2/M phase, NuMA, Cdk2 thr-160 and importin-α expression. These effects were prevented by catalase. These results demonstrate that H 2O 2 orchestrates a complex signaling network regulating S phase entry, nuclear trafficking and spindle pole formation through activation of Cdk2, importin-α, and NuMA. This pathway is essential for H 2O 2-induced mitosis in type II pneumocytes.

Preferential Expression of NuMA in the Nuclei of Proliferating Cells

Nuclear mitotic apparatus protein (NuMA) has an indispensable function in normal mitosis as an organizer of the mitotic spindle. NuMA is a prominent component of interphase cell nuclear matrix but its role during interphase is largely unknown. We examined the presence of NuMA in several human tissues. The majority of cells were positive for NuMA but a few negative cell types were found, including spermatozoa, superficial keratinocytes, neutrophil granulocytes, syncytiotrophoblasts, and some neurons, fibroblasts, and smooth and skeletal muscle cells. We further investigated the presence of NuMA in a cultured estrogen-dependent human breast cancer cell line and observed the disappearance of nuclear NuMA in the quiescent cells. The percentage of NuMA-positive cells diminished from an initial ∼100 to 60% during 6 days of culture. The presence of NuMA correlated positively with the presence of proliferation marker Ki-67 antigen and negatively with the culture time, confluence, and size of the cell islets. These results show that some nonproliferating, highly differentiated cell types lack NuMA and that cells may lose their NuMA without dramatic effects on the nuclear shape. This suggests that NuMA may be a nonessential component of the interphase nucleus.

The expression of the nuclear matrix proteins NuMA, topoisomerase II-α, and -β in bone and osseous cell culture: regulation by parathyroid hormone

Bone cells undergo changes in cell structure during phenotypic development. Parathyroid hormone (PTH) induces a change in osteoblast shape, a determinant of collagen expression. We hypothesize that alterations in bone cell shape reflect and direct gene expression as governed, in part, by nuclear organization. In this study, we determined whether the expression of nuclear matrix proteins that mediate nuclear architecture, NuMA, topoisomerase II (topo II)-α, and -β, were altered during osteoblast development and response to PTH in vivo. NuMA forms an interphase nuclear scaffold in some cells, the absence of which may accommodate alterations in nuclear organization necessary for specific functions. Topo II enzymes are expressed in bone cells; the α-isoform is specific to proliferating cells. We used immunohistochemistry and flow cytometry to determine whether NuMA is expressed in the primary spongiosa of the rat metaphyseal femur and whether expression of NuMA, topo II-α, and II-β changes during osteoblast development or with PTH treatment. NuMA and topo II-β were expressed in marrow cells, osteoblasts, osteocytes, and chondrocytes. These proteins were not detected in osteoclasts in vivo, but were observed in cultured cells. Bone marrow cells expressed topo II-α. All three proteins were expressed in cultures of rat osteoblast-like UMR-106 cells. PTH treatment downregulated the number of topo II-α-immunopositive cells, correlated with a decrease in S-phase cells, in both bone tissue and cell culture. We conclude that, in vivo, nuclear matrix composition is altered during bone cell development and that anabolic doses of PTH attenuate the proliferative capacity of osteogenic cells, in part, by targeting topo II-α expression.

Comparison of nuclear matrix proteins between EGFR-antisense transfected and untransfected glioblastoma cells.

The protein composition of the nuclear matrix is both tissue and cell type specific, and it undergoes changes with differentiation and transformation. In the present study, nuclear matrix proteins of EGFR-antisense transfected glioblastoma cell lines, U87 and U343, were compared with untransfected cell lines using two dimensional-gel electrophoresis. After EGFR-antisense transfection, the protein compositions of the nuclear matrices in both cell lines were different. Several nuclear proteins were only found in EGFR-antisense transfected cell lines. There was no difference in NuMA expression in the transfected and untransfected cell lines. These results suggest that EGFR-antisense reduced tumorigenicity on human glioblastoma cells by changing nuclear matrix protein compositions.

Parathyroid Hormone Regulates the Expression of the Nuclear Mitotic Apparatus Protein in the Osteoblast-like Cells, ROS 17/2.8

The parathyroid hormone (PTH) signaling pathways that effect changes in osteoblast gene expression also alter the organization of the cytoskeletal proteins. PTH regulates the expression of nucleoskeletal proteins, such as nuclear mitotic apparatus protein (NuMA) and topoisomerase II-α. NuMA is a structural component of the interphase nucleus and organizes the microtubules of the mitotic spindle during mitogenesis. We propose that PTH-induced alterations in osteoblast cytoarchitecture are accompanied by changes in osteoblast nuclear structure that contribute to changes in gene expression. We used immunofluorescence and confocal microscopy to determine the effect of PTH on the expression and nuclear distribution of NuMA in the rat osteosarcoma cell line, ROS 17/2.8. Cells were treated with PTH or vehicle, then fixed and stained with NuMA antibody. Optical sections of interphase naive cells revealed a diffuse distribution of NuMA, interspersed with speckles, in the central nuclear planes but not in nucleoli. During the metaphase and anaphase, NuMA localized at the mitotic spindle apparatus. The percentage of NuMA-immunopositive ROS 17/2.8 cells decreased with increasing confluence, but serum starvation did not attenuate NuMA expression. Cell density-dependent changes in cytoskeletal organization were observed in these cells. PTH treatment induced changes in cytoskeletal organization and increased the percentage of NuMA-immunopositive ROS 17/2.8 cells. These data suggest that PTH effects changes in osteoblast nuclear architecture by regulating NuMA, and that these alterations may be coupled to cytoskeletal organization.