LINC00467 facilitates osteosarcoma progression by sponging miR­217 to regulate KPNA4 expression.

Osteosarcoma (OS) is a musculoskeletal malignancy that originates from interstitial cells. An increasing number of studies have verified that long non­coding RNAs (lncRNAs) participate in the progression of numerous types of cancer. It has been reported that LINC00467 is a cancer­promoting gene in some types of cancer; however, the regulatory mechanism of LINC00467 in OS remains unknown. In the present study, reverse transcription-quantitative PCR was used to determine LINC00467 expression in OS tissues and cells. Additionally, the impact of LINC00467­knockdown on OS cell proliferation, migration and invasion was analyzed using Cell Counting Kit­8, colony formation and Transwell assays, as well as western blot analysis. RNA pulldown and luciferase reporter assays were conducted to investigate the regulatory mechanism of LINC00467 in OS. The results delineated that LINC00467 expression was elevated in OS tissues and cells, and that high LINC00467 expression was associated with a poor prognosis in patients with OS. LINC00467 inhibition suppressed OS progression by inhibiting cell proliferation, migration, invasion and epithelial­mesenchymal transition. LINC00467 served as a molecular sponge for microRNA (miR)­217, while karyopherin subunit α4 (KPNA4) was a downstream target gene of miR­217. Moreover, the overexpression of KPNA4 reversed the inhibitory effects of LINC00467 inhibition on OS progression. Therefore, the present study elucidated the potential mechanism of LINC00467 in OS and indicated that LINC00467 exerted its carcinogenic effects on OS through the miR­217/KPNA4 axis, implying that LINC00467 may be a novel potential therapeutic target for OS.

Cellular Importin-α3 Expression Dynamics in the Lung Regulate Antiviral Response Pathways against Influenza A Virus Infection.

Importin-α adaptor proteins orchestrate dynamic nuclear transport processes involved in cellular homeostasis. Here, we show that importin-α3, one of the main NF-κB transporters, is the most abundantly expressed classical nuclear transport factor in the mammalian respiratory tract. Importin-α3 promoter activity is regulated by TNF-α-induced NF-κB in a concentration-dependent manner. High-level TNF-α-inducing highly pathogenic avian influenza A viruses (HPAIVs) isolated from fatal human cases harboring human-type polymerase signatures (PB2 627K, 701N) significantly downregulate importin-α3 mRNA expression in primary lung cells. Importin-α3 depletion is restored upon back-mutating the HPAIV polymerase into an avian-type signature (PB2 627E, 701D) that can no longer induce high TNF-α levels. Importin-α3-deficient mice show reduced NF-κB-activated antiviral gene expression and increased influenza lethality. Thus, importin-α3 plays a key role in antiviral immunity against influenza. Lifting the bottleneck in importin-α3 availability in the lung might provide a new strategy to combat respiratory virus infections.

KPNA2/ERG Coexpression is Associated With Early Recurrence in Advanced Prostate Cancers.

Most prostate cancers (PC) overexpress the ERG oncogene and karyopherin α 2 (KPNA2). These genes play a role in prostatic carcinogenesis, but their prognostic significance is still debated. The aim of this study was to determine the prognostic significance of ERG and KPNA2 expression, and their association to early prostate-specific antigen (PSA) biochemical recurrence in advanced PC with lymph node metastases. A series of 65 consecutive pN1 M0 R0 PC samples obtained by radical prostatectomy with lymphadenectomy has been analyzed for ERG and KPNA2 expression by immunohistochemistry. For each case, the following clinical data were collected: age, preoperative serum PSA levels, Gleason grade group, TNM stage, and follow-up. PC recurrence was investigated by serum PSA assay and defined by a PSA concentration >0.2 ng/mL after a nadir of <0.1 ng/mL following radical prostatectomy. ERG-positive staining was found in 25/65 cases (38%), and KPNA2 in 56/65 cases (86%); neither was detected in normal prostatic tissue. Immunohistochemical concordance was found between primary tumor and lymph node metastases in 24/25 (96%) of ERG and 53/56 (95%) of KPNA2-positive cases. The follow-up was known in all cases, and early PSA recurrence occurred in 25/65 cases (38%). ERG positivity, both alone and in conjunction with KPNA2 positivity, was strongly associated with early PSA recurrence [both ERG+ and KPNA+, odds ratio: 22.2 (95% confidence interval, 6.0-82.3); ERG+ alone odds ratio: 17.9 (95% confidence interval, 5.1-63.5); P<0.0001 for both]. KPNA2 expression was significantly associated with the tumor stage (P<0.00001). The results suggest that the ERG+ phenotype might be selected in metastasis-initiating clones. ERG and KPNA2 may have a prognostic value, and their positivity in PC might warrant more aggressive treatments.

KPNA2 promotes metabolic reprogramming in glioblastomas by regulation of c-myc.

BACKGROUND: Cancer cells maintain energy metabolism mainly by glycolysis, even under sufficient oxygen conditions. It gives cancer cells better growth advantages under complicated internal environment. KPNA2 is a novel oncogene that has received much attention in recent years, but the exact mechanisms of KPNA2 in tumorigenesis and progression are largely unknown. Especially its potential roles in the metabolic transformation of tumors still remain to be explored. METHODS: The expressions of KPNA2 in glioblastoma and normal human brain samples were analyzed by immunohistochemical analysis. The activities of key enzymes in glycolysis, the production of lactate acid and glucose uptake were investigated by colorimetry. GLUT-1 expression was measured by flow cytometry. CCK8 was used to examine the cell viability in vitro, and the xenograft models in nude mice were established to explore the roles of KPNA2 in vivo. In addition, Co-IP, subcellular fractionation, western blot, immunofluorescence and luciferase assay were used to investigate the internal connection between KPNA2, c-myc and E2F1. RESULTS: In the present study, we found that KPNA2 was highly expressed in the glioma compared to the normal brain tissues. Level of KPNA2 was an independent predictor of prognosis in the glioma patients. Knockdown of KPNA2 in the glioblastoma cell lines U87 and U251 decreased deoxyglucose uptake, activities of the key glycolytic enzymes and lactate production. The level of oxidative phosphorylation (OXPHOS) was moderately decreased. Additioanlly, tumor proliferation and invasiveness were concomitantly downregulated. We have identified c-myc as a potential mediator of KPNA2. Aberrant expression of KPNA2 significantly changed the subcellular distribution of c-myc as well as its expression level. E2F1, another key cargo protein of KPNA2, was further identified to play a potential role in regulating the transcription of c-myc by KPNA2. CONCLUSIONS: Our findings suggested that KPNA2, a potential tumor oncogene, performs its function in part via regulating cellular metabolism through c-myc signaling axis. It would provide a possible explanation for Warburg effect and thus offer a new perspective to the roles of KPNA2 in gliomagenesis.

MicroRNA-302 Cluster Downregulates Enterovirus 71-Induced Innate Immune Response by Targeting KPNA2.

Enterovirus 71 (EV71) induces significantly elevated levels of cytokines and chemokines, leading to local or systemic inflammation and severe complications. As shown in our previous study, microRNA (miR) 302c regulates influenza A virus-induced IFN expression by targeting NF-κB-inducing kinase. However, little is known about the role of the miR-302 cluster in EV71-mediated proinflammatory responses. In this study, we found that the miR-302 cluster controls EV71-induced cytokine expression. Further studies demonstrated that karyopherin α2 (KPNA2) is a direct target of the miR-302 cluster. Interestingly, we also found that EV71 infection upregulates KPNA2 expression by downregulating miR-302 cluster expression. Upon investigating the mechanisms behind this event, we found that KPNA2 intracellularly associates with JNK1/JNK2 and p38, leading to translocation of those transcription factors from the cytosol into the nucleus. In EV71-infected patients, miR-302 cluster expression was downregulated and KPNA2 expression was upregulated compared with controls, and their expression levels were closely correlated. Taken together, our work establishes a link between the miR-302/ KPNA2 axis and EV71-induced cytokine expression and represents a promising target for future antiviral therapy.

Karyopherin α-2 is a reliable marker for identification of patients with high-risk stage II colorectal cancer.

PURPOSE: Adjuvant chemotherapy (AC) is frequently considered in patients with high-risk stage II colorectal cancer (CRC). Among patients with stage II CRC who do not receive AC because they are not considered to be at high risk, 20-25% will develop recurrence and die from the disease. Elevated levels of KPNA2 have been observed in various cancers, and overexpression of KPNA2 is related to CRC progression. METHODS: We examined the expression of KPNA2 using 293 CRC tissues, including 118 with stage II CRC, and investigated the applicability of KPNA2 as a biomarker to predict high-risk stage II CRC. Moreover, we further investigated the role of KPNA2 as an oncogene in CRC carcinogenesis using in vitro functional studies. RESULTS: High KPNA2 expression was associated with vascular (p = 0.027) and lymphatic invasion (p = 0.009) in patients with stage II CRC. On multivariate analysis, high KPNA2 expression (HR 3.174, 95% CI 2.060-4.889; p < 0.001) was independently associated with survival in patients with CRC. The overall survival rate in patients with high KPNA2 expression was higher than that in patients with low KPNA2 expression in CRC (p < 0.001), even in patients with stage II CRC (p = 0.001). Additionally, KPNA2 was associated with tumorigenesis and cancer progression in CRC cells; high KPNA2 expression was associated with increased cell proliferation (p < 0.05), migration (p = 0.03), invasion (p = 0.001), and semisolid agar colony formation (p < 0.001). CONCLUSION: KPNA2 expression is useful for identification of patients with high-risk stage II CRC who could benefit from AC and that KPNA2 may also be a promising therapeutic target.

Expression of importin-α isoforms in human nasal mucosa: implication for adaptation of avian influenza A viruses to human host.

BACKGROUND: Transportation into the host cell nucleus is crucial for replication and transcription of influenza virus. The classical nuclear import is regulated by specific cellular factor, importin-α. Seven isoforms of importin-α have been identified in human. The preference of importin-α3 of avian influenza virus and -α7 isoform of human strains during replication in human cells was previously identified. In addition, both avian and human influenza viruses were shown to use importin-α1 isoform for their replication. FINDING: The mRNA levels of importin-α1, -α3, and -α7 isoforms in human respiratory tract was determined by real-time RT-PCR. The results indicate that mRNA level of importin-α7 was significantly higher than that of importin-α1 (p-value < 0.0001) and importin-α3 (p-value < 0.0001) isoforms in human nasal mucosa while importin-α1 was detected as the highest expression importin-α isoform in lung tissues. CONCLUSIONS: These results may explain the preference of importin-α7 isoforms in seasonal influenza viruses in human upper respiratory tract and may suggest a selective pressure toward importin-α7 in human respiratory tract infection of an avian virus.

mTOR regulates proteasomal degradation and Dp1/E2F1- mediated transcription of KPNA2 in lung cancer cells.

Karyopherin subunit alpha-2 (KPNA2) is overexpressed in various human cancers and is associated with cancer invasiveness and poor prognosis in patient. Nevertheless, the regulation of KPNA2 expression in cancers remains unclear. We herein applied epidermal growth factor (EGF) and five EGF receptor (EGFR)-related kinase inhibitors to investigate the role of EGFR signaling in KPNA2 expression in non-small cell lung cancer (NSCLC) cells. We found that EGFR signaling, particularly the mammalian target of rapamycin (mTOR) activity was positively correlated with KPNA2 protein levels in NSCLC cells. The mTOR inhibitors and mTOR knockdown reduced the protein and mRNA levels of KPNA2 in NSCLC and breast cancer cells. Specifically, rapamycin treatment induced proteasome-mediated KPNA2 protein decay and attenuated the transcriptional activation of KPNA2 by decreasing Dp1/E2F1 level in vivo. Immunoprecipitation assay further revealed that KPNA2 physically associated with the phospho-mTOR/mTOR and this association was abolished by rapamycin treatment. Collectively, our results show for the first time that KPNA2 is transcriptionally and post-translationally regulated by the mTOR pathway and provide new insights into targeted therapy for NSCLC.

DnaJ-1 and karyopherin α3 suppress degeneration in a new Drosophila model of Spinocerebellar Ataxia Type 6.

Spinocerebellar ataxia type 6 (SCA6) belongs to the family of CAG/polyglutamine (polyQ)-dependent neurodegenerative disorders. SCA6 is caused by abnormal expansion in a CAG trinucleotide repeat within exon 47 of CACNA1A, a bicistronic gene that encodes α1A, a P/Q-type calcium channel subunit and a C-terminal protein, termed α1ACT. Expansion of the CAG/polyQ region of CACNA1A occurs within α1ACT and leads to ataxia. There are few animal models of SCA6. Here, we describe the generation and characterization of the first Drosophila melanogaster models of SCA6, which express the entire human α1ACT protein with a normal or expanded polyQ. The polyQ-expanded version of α1ACT recapitulates the progressively degenerative nature of SCA6 when expressed in various fly tissues and the presence of densely staining aggregates. Additional studies identify the co-chaperone DnaJ-1 as a potential therapeutic target for SCA6. Expression of DnaJ-1 potently suppresses α1ACT-dependent degeneration and lethality, concomitant with decreased aggregation and reduced nuclear localization of the pathogenic protein. Mutating the nuclear importer karyopherin α3 also leads to reduced toxicity from pathogenic α1ACT. Little is known about the steps leading to degeneration in SCA6 and the means to protect neurons in this disease are lacking. Invertebrate animal models of SCA6 can expand our understanding of molecular sequelae related to degeneration in this disorder and lead to the rapid identification of cellular components that can be targeted to treat it.

Gene expression analysis for evaluation of potential biomarkers in hepatocellular carcinoma.

BACKGROUND: The poor prognosis of hepatocellular carcinoma (HCC) and the lack of specific screening markers underline the need for new biomarkers for human hepatocarcinogenesis. MATERIALS AND METHODS: We investigated 10 postulated biomarkers for HCC (AFP, GPC3, OPN, IGF1, HGF, SPINK1, KPNA, FUCA1, CgA, HSP90) with microarray gene expression analysis and real-time polymerase chain reaction (RT-PCR) in HCC tissues of different etiologies. RESULTS: Four candidate genes (FUCA1, HGF, IGF1, CgA) showed low median fold changes (fc) of expression compared to corresponding non-malignant liver tissues (fc range=0.2-0.8; maximum 15% of samples). The classic biomarker, alpha-fetoprotein (AFP), was significantly over-expressed (fc=2.4) in 30% of tumors. High tumor AFP expression was associated with significantly elevated serum AFP concentrations (>90% of cases). Five genes (OPN, SPINK1, GPC3, HSP90, KNPA2) showed significantly higher expression than AFP in 64% to 82% of samples (median fc range=2.9-8.3). RT-PCR analyses gave similar results. CONCLUSION: Unlike previous studies, our results did not confirm FUCA1, HGF, IGF1 or CgA as potential markers for HCC. In contrast, OPN, SPINK1, GPC3 and KNPA2 were significantly over-expressed in HCC tissues. These genes may be useful in developing future biomarkers and therapeutic strategies for HCC.

Characterization of the nuclear import mechanism of the CCAAT-regulatory subunit Php4.

Php4 is a nucleo-cytoplasmic shuttling protein that accumulates in the nucleus during iron deficiency. When present in the nucleus, Php4 associates with the CCAAT-binding protein complex and represses genes encoding iron-using proteins. Here, we show that nuclear import of Php4 is independent of the other subunits of the CCAAT-binding complex. Php4 nuclear import relies on two functionally independent nuclear localization sequences (NLSs) that are located between amino acid residues 171 to 174 (KRIR) and 234 to 240 (KSVKRVR). Specific substitutions of basic amino acid residues to alanines within these sequences are sufficient to abrogate nuclear targeting of Php4. The two NLSs are biologically redundant and are sufficient to target a heterologous reporter protein to the nucleus. Under low-iron conditions, a functional GFP-Php4 protein is only partly targeted to the nucleus in imp1Δ and sal3Δ mutant cells. We further found that cells expressing a temperature-sensitive mutation in cut15 exhibit increased cytosolic accumulation of Php4 at the nonpermissive temperature. Further analysis by pull-down experiments revealed that Php4 is a cargo of the karyopherins Imp1, Cut15 and Sal3. Collectively, these results indicate that Php4 can be bound by distinct karyopherins, connecting it into more than one nuclear import pathway.

The prognostic impact of high Nijmegen breakage syndrome (NBS1) gene expression in ERG-negative prostate cancers lacking PTEN deletion is driven by KPNA2 expression.

The Nijmegen breakage syndrome (NBS1) gene was suggested as a prostate cancer susceptibility gene. This study was undertaken to determine, whether NBS1 expression is linked to clinically or molecularly relevant subgroups of prostate cancer. NBS1 expression was analyzed by immunohistochemistry on a tissue microarray containing 11,152 prostate cancer specimens. NBS1 expression was absent or only weakly detectable in benign prostate. In prostate cancers, NBS1 expression was found in 81.3% of interpretable tumors and was considered strong in 41.3% of cases. NBS1 upregulation was tightly linked to ERG-positive cancers (p<0.0001). Within ERG-negative cancers, strong NBS1 immunostaining was linked to advanced pathological tumor stage, high Gleason grade, and positive nodal status (p<0.0001 each), while high NBS1 immunostaining was only weakly associated with advanced pathological tumor stage in ERG-positive cancers (p=0.0099). A comparison with chromosomal deletions revealed a strong NBS1 upregulation in PTEN-deleted cancers, while deletions of 3p13, 5q21 and 6q15 did not affect NBS1 expression. High NBS1 expression was linked to biochemical recurrence in ERG-negative and PTEN non-deleted cancers (p<0.0001), which was largely driven by high KPNA2 karyopherin alpha 2 expression. In conclusion, our study identifies an association of NBS1 expression with surrogates of genomic instability in prostate cancer including TMPRSS2-ERG rearrangements and PTEN deletion. The prognostic impact of NBS1 expression in ERG-negative, PTEN non-deleted cancers was dependent of the expression status of its interaction partner KPNA2.

Nuclear karyopherin a2: a novel biomarker for infiltrative astrocytomas.

The karyopherin (KPNA) protein family is involved in nucleocytoplasmic trafficking. Increased KPNA levels have been found to predict poor prognosis for a variety of solid tumors, including breast, ovarian, cervical, and prostate cancer, and melanoma. The purpose of this study was to evaluate karyopherin a2 as novel biomarker for astrocytic gliomas of WHO grades II-IV. We semiquantitatively measured nuclear expression of karyopherin a2 and the MIB1 labeling index, by immunohistochemical analysis, for 94 primary (23 astrocytomas WHO grade II, 24 astrocytomas WHO grade III, 47 glioblastomas) and 12 recurrent gliomas. In addition, IDH1 mutation status and Nijmegen breakage syndrome 1 protein expression were assessed, by immunohistochemical analysis, for all 71 malignant (WHO grade III and IV) and all 94 primary gliomas, respectively. Statistical analysis was performed by use of standard techniques. Karyopherin a2 expression correlated significantly with histological grade (p < 0.001), with proliferative activity as assessed by the MIB1 index (p < 0.001), with IDH1 mutation status (p = 0.032), and with Nijmegen breakage syndrome 1 protein expression (p = 0.001). Recurrent tumors expressed significantly higher levels of karyopherin a2 (p = 0.045) than primary growths. Multivariate analysis of the overall series identified low karyopherin a2 expression (defined as less than 5 %) as an independent prognostic predictor of overall (p = 0.041) and progression-free survival (p = 0.004). Survival of glioblastoma patients >5 years was seen only in those with KPNA2 expression levels ≤1 % (p = 0.014). KPNA2 expression may have potential as a novel diagnostic and prognostic biomarker for astrocytic gliomas.

Distinct effects of importin α2 and α4 on Oct3/4 localization and expression in mouse embryonic stem cells.

The cellular repertoire of importin (IMP) proteins that mediates nuclear import of transcription factors and chromatin remodeling agents is critical to processes such as differentiation and transformation. This study identifies for the first time independent roles for specific IMPαs in murine embryonic stem cells (mESCs), showing that mESC differentiation is accompanied by dynamic changes in the levels of transcripts encoding the IMPs, IMPα3, IMPα4, IMPß1, and IPO5. Of these, only IMPα4 was maintained at higher levels in differentiating mESCs, correlating with the finding that IMPα4 overexpression induced a significant decrease in Oct3/4 protein levels compared to control transfections. In parallel, IMPα4 protein showed a unique and striking shift in subcellular localization from the nucleus to the cytoplasm during differentiation, which is consistent with activation of a role in nuclear import of differentiation factors. Overexpression of a dominant-negative IMPα2 isoform, when assessed against adjacent untransfected or IMPα2 transfected cells, led to both a significant reduction in endogenous Oct3/4 protein levels and inhibition of Oct3/4 nuclear localization, suggesting that IMPα2-mediated delivery of Oct3/4 to the nucleus contributes directly to maintenance of mESC pluripotency. These findings implicate IMPα2 and IMPα4 in specific but distinct roles in the fate choice between pluripotency and commitment to differentiation.

Significance of karyopherin-{alpha} 2 (KPNA2) expression in esophageal squamous cell carcinoma.

BACKGROUND: Karyopherin-alpha 2 (KPNA2) is a member of the importin alpha family and has recently been reported to play an important role in tumorigenesis and tumor progression. The aim of the current study was to elucidate the clinicopathological significance of immunohistochemical expression of KPNA2 in esophageal squamous cell carcinoma (ESCC). PATIENTS AND METHODS: KPNA2 expression was investigated by immunohistochemistry in 116 surgically resected ESCC, and the association of KPNA2 expression with clinicopathologic features was also examined. RESULTS: Sixty (51.7%) ESCCs demonstrated positive expression of KPNA2. Positive expression of KPNA2 showed a significant association with poor differentiation (p=0.015), tumor depth (p=0.001), lymphatic invasion (p<0.001), venous invasion (p<0.001), and tumor stage (p=0.008). Positive expression of KPNA2 was also significantly associated with Ki-67 labeling index (p=0.039). Univariate analysis revealed that the prognosis of the ESCC patients whose tumors demonstrated positive expression of KPNA2 was significantly poorer than that of those that did not (p=0.009). Multivariate analysis revealed that only tumor depth and the presence of lymph node metastasis, which are strong prognostic factors in ESCC, were independently associated with poor prognosis in this study. CONCLUSION: KPNA2 expression is associated with poor differentiation, tumor invasiveness, and tumor proliferation in ESCC.

Nuclear karyopherin alpha2 expression predicts poor survival in patients with advanced breast cancer irrespective of treatment intensity.

Intensive lymph node involvement indicates poor prognosis in breast cancer patients. The significance of other molecular prognostic factors in this subgroup is unclear. Karyopherin alpha2 (KPNA2) has been reported as an important factor of tumorgenesis and progression of breast cancer. The aim of present study was to evaluate the impact of KPNA2 expression on prognosis of patients with high risk breast cancer (HRBC) and response intensive chemotherapy within the randomized WSG-AM-01 trial. KPNA2 nuclear expression (>10% vs. <10% of nuclei) was measured by immunohistochemistry on tissue arrays of 191 patients randomized to tandem high dose vs. conventional dose-dense chemotherapy in HRBC with >9 positive lymph nodes and correlated with clinical outcome (median follow-up of 63.3 months) by Kaplan-Meier and multivariate Cox hazard model analysis, including, molecular subtypes determined by k-clustering (k = 5). KPNA2 overexpression (n = 74, 39%) significantly correlated with shorter event-free and overall survival (OS) in both therapy arms by univariate analysis. Multivariate analysis showed that the overexpression of KPNA2 was an independent prognostic factor of decreased OS HR = 1.86 [95% CI: 1.07-3.23, p = 0.03]. This predictive value was independent of basal-like/Her-2/neu subtypes, significantly associated with KPNA2 and was addressed particularly to G2 tumors. Our data suggest the use of KPNA2 nuclear expression as novel prognostic marker in node-positive patients, especially in determination of G2 tumors in 2 subgroups of different prognosis. KPNA2 expression may be also considered as a marker for global chemoresistance, which can not be overcome by conventional dose-modification of chemotherapy in advanced breast cancer.

KPNA2 protein expression in invasive breast carcinoma and matched peritumoral ductal carcinoma in situ.

The aim of this study was to evaluate protein expression of Karyopherin alpha 2 (KPNA2) in invasive breast cancer and matched ductal carcinoma in situ (DCIS) and to correlate it with clinicopathological data, including patient survival. KPNA2 protein expression was assessed by immunohistochemistry in breast tissue samples, containing invasive carcinomas, DCIS, and adjacent histologically benign breast tissues. A polyclonal goat KPNA2 antibody was used for immunostaining of 83 clinicopathologically characterized cases. For statistical analysis, staining of at least 10% of nuclei was considered KPNA2 positive. Immunohistochemical detection of KPNA2 in invasive carcinoma showed a significant correlation with higher tumor stage, positive lymph node status, higher tumor grade, and negative ER status. Concordantly, KPNA2-positive tumors (31.3%) showed significantly shorter disease-free survival times (69 months vs 118 months; p = 0.007). KPNA2 protein expression was also detected in DCIS (21.3%) adjacent to invasive tumor and correlated with nuclear grade (p = 0.013). Expression of KPNA2 in invasive breast cancer correlates with conventional prognostic parameters and shorter disease-free survival. Additionally, KPNA2 is overexpressed in DCIS, particularly high grade lesions, which emphasizes its potential role in carcinogenesis of invasive breast carcinomas.

Novel nuclear import of Vpr promoted by importin alpha is crucial for human immunodeficiency virus type 1 replication in macrophages.

Monocytes/macrophages are major targets of human immunodeficiency virus type 1 (HIV-1) infection. The viral preintegration complex (PIC) of HIV-1 enters the nuclei of monocyte-derived macrophages, but very little PIC migrates into the nuclei of immature monocytes. Vpr, one of the accessory gene products of HIV-1, is essential for the nuclear import of PIC in these cells, although the role of Vpr in the entry mechanism of PIC remains to be clarified. We have shown previously that Vpr is targeted to the nuclear envelope and then transported into the nucleus by importin alpha alone, in an importin beta-independent manner. Here we demonstrate that the nuclear import of Vpr is strongly promoted by the addition of cytoplasmic extract from macrophages but not of that from monocytes and that the nuclear import activity is lost with immunodepletion of importin alpha from the cytoplasmic extract. Immunoblot analysis and real-time PCR demonstrate that immature monocytes express importin alpha at low levels, whereas the expression of three major importin alpha isoforms markedly increases upon their differentiation into macrophages, indicating that the expression of importin alpha is required for nuclear import of Vpr. Furthermore, interaction between importin alpha and the N-terminal alpha-helical domain of Vpr is indispensable, not only for the nuclear import of Vpr but also for HIV-1 replication in macrophages. This study suggests the possibility that the binding of Vpr to importin alpha, preceding a novel nuclear import process, is a potential target for therapeutic intervention.

Importin alpha mRNAs have distinct expression profiles during spermatogenesis.

Importin proteins control access to the cell nucleus by mediating the nuclear transport of specific cargoes. We hypothesized that developmental regulation of gene expression may be partially effected by changes in the nuclear transport machinery complement, manifested as regulated expression of importin alpha family genes. We first clarified the identity of the five known mouse importin alpha genes relative to those for human and then determined their expression throughout postnatal rodent testis using PCR and in situ hybridization. Distinct expression patterns were observed for each. At 10 dpp, all importin alpha mRNAs were detected in spermatogonia. In the adult mouse testis, importins alpha1 and alpha3 were detected in spermatogonia and early pachytene spermatocytes. Importin alpha4 mRNA was identified in pachytene spermatocytes, alpha6 mRNA in round spermatids, and alpha2 mRNA in both of these. The distinct importin alpha expression patterns are consistent with their having specific roles and transport cargoes during spermatogenesis.

Patterns of importin-alpha expression during Drosophila spermatogenesis.

Importin-alpha proteins do not only mediate the nuclear import of karyophilic proteins but also regulate spindle assembly during mitosis and the assembly of ring canals during Drosophila oogenesis. Three importin-alpha genes are present in the genome of Drosophila. To gain further insights into their function we analysed their expression during spermatogenesis by using antibodies raised against each of the three Importin-alpha proteins identified in Drosophila, namely, Imp-alpha1, -alpha2, and -alpha3. We found that each Imp-alpha is expressed during a specific and limited period of spermatogenesis. Strong expression of Imp-alpha2 takes place in spermatogonial cells, persists in spermatocytes, and lasts up to the completion of meiosis. In growing spermatocytes, the intracellular localisation of Imp-alpha2 appears to be dependent upon the rate of cell growth. In pupal testes Imp-alpha2 is essentially present in the spermatocyte nucleus but is localised in the cytoplasm of spermatocytes from adult testes. Both Imp-alpha1 and -alpha3 expression initiates at the beginning of meiosis and ends during spermatid differentiation. Imp-alpha1 expression extends up to the onset of the elongation phase, whereas that of Imp-alpha3 persists up to the completion of nuclear condensation when the spermatids become individualised. During meiosis Imp-alpha1 and -alpha3 are dispersed in the karyoplasm where they are partially associated with the nuclear spindle, albeit not with the asters. At telophase they aggregate around the chromatin. During sperm head differentiation, both Imp-alpha1 and -alpha3 are nuclear. These data indicate that each Imp-alpha protein carries during Drosophila spermatogenesis distinct, albeit overlapping, functions that may involve nuclear import of proteins, microtubule organisation, and other yet unknown processes.