The proper orientation of the mitotic spindle is essential for mitosis; however, how these events unfold at the molecular level is not well understood. AMP-activated protein kinase (AMPK) regulates energy homeostasis in eukaryotes, and AMPK-null Drosophila mutants have spindle defects. We show that threonine 172 phosphorylated AMPK localizes to the mitotic spindle poles and increases when cells enter mitosis. AMPK depletion causes a mitotic delay with misoriented spindles relative to the normal division plane and a reduced number and length of astral microtubules. AMPK-depleted cells contain mitotic actin bundles, which prevent astral microtubule-actin cortex attachments. Precise control of the cell division plane is achieved through the proper assembly, positioning, and orientation of the microtubule-based spindle. In nonpolarized adherent cells, the spindle orients parallel to the substratum (reviewed in reference 14) and positions itself centrally to ensure an accurate distribution of genetic information and an equal composition of daughter cells (22,25,44). When spindles are misoriented, daughter cell placement in tissue is abnormal, potentially leading to tissue disorganization and cancer metastasis (24). Though some of the major components of the spindle (e.g., microtubules and motor proteins) have been intensely studied in spindle orientation, the molecular signaling pathways regulating these events have not been well established.Astral microtubules emanating from the spindle poles attach to the actin cortex and are essential for proper spindle orientation (6, 8); however, recently it has appeared that the establishment and maintenance of spindle orientation and positioning are more complex than previously believed and involve multiple pathways. The PtdIns-(3,4,5)P3 direct dynein/dynactin forces to orient the spindle parallel to the substratum, a process overseen by the small Rho GTPase cdc42 (35). Transmembrane integrins are essential for spindle orientation by maintaining substrate adhesion contacts during mitosis (36, 37). Actin itself also serves multiple functions that go beyond its role in the cortex, whereby F-actin forms dynamic cables encaging the spindle to function in spindle anchoring and length (48). Furthermore, actin-binding proteins orient and assemble the microtubule spindle. For instance, myosin 10, which localizes to mitotic spindle poles, is required for proper spindle anchoring and length (48), and moesin is required for spindle symmetry and positioning (15). Thus, spindle orientation and positioning are overseen by a complex interplay of signaling proteins, microtubules and associated proteins, and actin and associated proteins.AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine kinase that consists of a catalytic ␣ subunit and regulatory ␤ and ␥ subunits (33, 47). AMPK regulates energy homeostasis in all eukaryotic organisms and is active when ADP levels are high and ATP levels are low (9). AMPK activity is regulated by phosphorylation at AMPK thr172 (pAMPK thr...

The role of hepatocyte nuclear factor 1␣ (HNF1␣) in the regulation of hepatitis B virus (HBV) transcription and replication in vivo was investigated using a HNF1␣-null HBV transgenic mouse model. HBV transcription was not measurably affected by the absence of the HNF1␣ transcription factor. However, intracellular viral replication intermediates were increased two-to fourfold in mice lacking functional HNF1␣ protein. The increase in encapsidated cytoplasmic replication intermediates in HNF1␣-null HBV transgenic mice was associated with the appearance of nonencapsidated nuclear covalently closed circular (CCC) viral genomic DNA. Viral CCC DNA was not readily detected in HNF1␣-expressing HBV transgenic mice. This indicates the synthesis of nuclear HBV CCC DNA, the proposed viral transcriptional template found in natural infection, is regulated either by subtle alterations in the levels of viral transcripts or by changes in the physiological state of the hepatocyte in this in vivo model of HBV replication.Hepatitis B virus (HBV) is an enveloped virus that infects the livers of humans and other primates (1,20). In infected hepatocytes, the 3.2-kb DNA genome is transcribed by the cellular RNA polymerase II, generating the 3.5-, 2.4-, 2.1-, and 0.7-kb viral RNAs (7). These transcripts encode the nucleocapsid polypeptides, the large surface antigen polypeptide, the middle and major surface antigen polypeptides, and the Xgene polypeptide, respectively (7). In addition, the 3.5-kb pregenomic RNA encodes the viral polymerase and is reverse transcribed by this polypeptide within the viral nucleocapsid to produce the 3.2-kb viral genomic DNA (16). The mature nucleocapsids containing viral genomic DNA can be secreted from the cell in virus particles only by associating with surface antigen polypeptides within the membrane of the endoplasmic reticulum (2,3,8). Virus buds into the lumen of the endoplasmic reticulum and is transported out of the cell through the Golgi apparatus (12, 28). The ability of the mature nucleocapsid to form virus particles depends on the correct level of synthesis of the surface antigen polypeptides (2). In the absence of surface antigen polypeptide synthesis, the mature nucleocapsids would be expected to transport the viral genome back into the nucleus, where the partially double-stranded viral genome is converted into covalently closed circular (CCC) DNA that represents the proposed viral transcriptional template. Therefore, the level of transcription of the 2.4-and 2.1-kb viral RNAs, in addition to the 3.5-kb pregenomic RNA, is likely to influence viral replication in general and nuclear HBV CCC DNA accumulation in particular.As the relative abundance of mature nucleocapsids and surface antigen polypeptides is likely to be influenced by the levels of their corresponding RNAs, the regulation of the level of viral transcription is expected to influence directly viral replication. Using transient transfection analysis in various cell culture systems, it has been demonstrated that the transcription of t...

Acetylated tubulin (AT) expression has been proposed as a marker for sensitivity to taxane chemotherapy. We wanted to explore AT as a prognostic marker in squamous cell carcinoma of the head and neck (SCCHN). We assessed AT expression in archival tissue from our institutional tissue bank of primary SCCHN specimens. We also examined AT expression on pre-therapy tissues of patients with SCCHN receiving induction chemotherapy with docetaxel, cisplatin and 5FU (TPF IC). AT expression was assessed on archival cases of SCCHN with (N = 63) and without (N = 82) locoregional lymph node metastases (LNM). The predominant tumor site was oral cavity (52 %). Immunohistochemistry staining was based on staining intensity and percentage of tumor cells stained to create a weighted index (WI). A total of nine patients who received TPF IC were evaluable for response by RECIST and also had pre-therapy tissues available. A significant independent correlation between AT and tumor grade (p = 0.001) and primary location (p = 0.008) was noted. There was a trend of higher AT in patients with presence of LNM (p = 0.052) and a trend in improved OS for patients with an AT WI below the median compared to those above the median for patients with no LNM (p = 0.054). For patients treated with induction TPF, we observed an inverse correlation between AT expression and response to TPF IC (p = 0.0071). AT expression is correlated with tumor grade and primary site. There was an observed trend correlating AT with presence nodal metastases. The observed inverse correlation with response to taxane based chemotherapy needs validation in a larger sample size.

Background: STRAD␣ is the cofactor of the tumor suppressor LKB1; however, it is unclear if STRAD␣ has LKB1-independent roles. Results: STRAD␣ complexes with the kinase PAK1 to modify PAK1 phosphorylation likely via rac1 and control cell motility when LKB1 is null. Conclusion: STRAD␣ regulates PAK1 in LKB1-null cells to oversee cancer cell polarity and invasion. Significance: This shows an undiscovered role of STRAD␣ distinct from the LKB1 pathway.

The role of the peroxisome proliferator-activated receptor α (PPARα) in regulating hepatitis B virus (HBV) transcription and replication in vivo was investigated in an HBV transgenic mouse model. Treatment of HBV transgenic mice with the peroxisome proliferators Wy-14,643 and clofibric acid resulted in a less than twofold increase in HBV transcription rates and steady-state levels of HBV RNAs in the livers of these mice. In male mice, this increase in transcription was associated with a 2- to 3-fold increase in replication intermediates, whereas in female mice it was associated with a 7- to 14-fold increase in replication intermediates. The observed increases in transcription and replication were dependent on PPARα. HBV transgenic mice lacking this nuclear hormone receptor showed similar levels of HBV transcripts and replication intermediates as untreated HBV transgenic mice expressing PPARα but failed to demonstrate alterations in either RNA or DNA synthesis in response to peroxisome proliferators. Therefore, it appears that very modest alterations in transcription can, under certain circumstances, result in relatively large increases in HBV replication in HBV transgenic mice.