Long-term outcomes of whole-pelvis radiation therapy using volumetric modulated arc therapy for high-risk prostate cancer†.

This study investigated the outcomes of whole-pelvis radiation therapy (WPRT) using volumetric modulated arc therapy (VMAT) for high-risk prostate cancer. We retrospectively analysed 112 patients with high-risk prostate cancer who started WPRT at our hospital between August 2011 and August 2015. The prescribed dose was 78 Gy in 39 fractions to the prostate and 46.8 Gy in 26 fractions to the pelvic lymph node (LN) area. All patients received long-term androgen deprivation therapy. We evaluated late gastrointestinal (GI) and genitourinary (GU) toxicities using the Common Terminology Criteria for Adverse Events version 4.0. The median follow-up period for censored cases was 97 (interquartile range [IQR] = 85-108) months. The median age was 72 (IQR = 67-75) years. The high-risk and very-high-risk groups included 41 (36.6%) and 71 patients (63.4%), respectively. The median risk of LN invasion calculated by the Roach formula was 36.9 (IQR = 26.6-56.3) %. The 8-year overall survival, biochemical failure-free survival, disease-free survival and distant metastasis-free survival rates were 88.4, 91.9, 83.8 and 98.0%, respectively. Only one patient experienced common iliac LN recurrence, which was outside the pelvic irradiation area. All patients with recurrent disease were categorized into the very-high-risk group. The 8-year cumulative rates of ≥Grade 2 late GI and GU toxicities were 12.8 and 11.8%, respectively. No patients experienced Grade 4 or higher toxicities. WPRT using VMAT for high-risk prostate cancer was well tolerated and effective.

Comparison of Late Toxicity After Whole-pelvis Versus Prostate-only VMAT for Prostate Cancer.

BACKGROUND/AIM: To evaluate whether whole-pelvis (WP) volumetric modulated arc therapy (VMAT) is associated with increased late toxicity compared with prostate-only (PO) VMAT in patients with localized prostate cancer. PATIENTS AND METHODS: Participants comprised 384 consecutive patients treated with definitive VMAT to 78 Gy in 39 fractions from July 2011 to August 2016. Of these, 183 patients received PO-VMAT and 201 patients received initial WP-VMAT to 46.8 Gy in 26 fractions using a simultaneous integrated boost technique. Gastrointestinal (GI) and genitourinary (GU) toxicities were prospectively scored using Common Terminology Criteria for Adverse Events version 4.0. RESULTS: Median follow-up was 49 months (range=16-88 months) in the PO-VMAT group and 52 months (range=10-85 months) in the WP-VMAT group. Frequencies of Grade 3 late GI and GU toxicities were ≤3% across both groups. No patients experienced Grade 4+ toxicity. Cumulative incidences of Grade 2+ late GI and GU toxicities were similar between PO- and WP-VMAT groups (p=0.508 and p=0.838, respectively). Five-year cumulative incidences of Grade 2+ late GI and GU toxicities were 12.2% and 6.6% for the PO-VMAT group and 12.3% and 8.9% for the WP-VMAT group, respectively. CONCLUSION: WP-VMAT did not increase late GI and GU toxicities. This suggests that concerns about increasing toxicity profile are insufficient reason for omitting WPRT for patients with high-risk prostate cancer.

Multiple factors in the early splicing complex are involved in the nuclear retention of pre-mRNAs in mammalian cells.

Intron-containing pre-mRNAs are retained in the nucleus until they are spliced. This mechanism is essential for proper gene expression. Although the formation of splicing complexes on pre-mRNAs is thought to be responsible for this nuclear retention activity, the details are poorly understood. In mammalian cells, in particular, very little information is available regarding the retention factors. Using a model reporter gene, we show here that U1 snRNP and U2AF but not U2 snRNP are essential for the nuclear retention of pre-mRNAs in mammalian cells, showing that E complex is the major entity responsible for the nuclear retention of pre-mRNAs in mammalian cells. By focusing on factors that bind to the 3'-splice site region, we found that the 65-kD subunit of U2AF (U2AF(65) ) is important for nuclear retention and that its multiple domains have nuclear retention activity per se. We also provide evidence that UAP56, a DExD-box RNA helicase involved in both RNA splicing and export, cooperates with U2AF(65) in exerting nuclear retention activity. Our findings provide new information regarding the pre-mRNA nuclear retention factors in mammalian cells.

Molecular determinants of substrate recognition in thermostable alpha-glucosidases belonging to glycoside hydrolase family 13.

Bacillus stearothermophilus alpha-1,4-glucosidase (BS) is highly specific for alpha-1,4-glucosidic bonds of maltose, maltooligosaccharides and alpha-glucans. Bacillus thermoglucosdasius oligo-1,6-glucosidase (BT) can specifically hydrolyse alpha-1,6 bonds of isomaltose, isomaltooligosaccharides and alpha-limit dextrin. The two enzymes have high homology in primary structure and belong to glycoside hydrolase family 13, which contain four conservative regions (I, II, III and IV). The two enzymes are suggested to be very close in structure, even though there are strict differences in their substrate specificities. Molecular determinants of substrate recognition in these two enzymes were analysed by site-directed mutagenesis. Twenty BT-based mutants and three BS-based mutants were constructed and characterized. Double substitutions in BT of Val200 -->Ala in region II and Pro258 -->Asn in region III caused an appearance of maltase activity compared with BS, and a large reduction of isomaltase activity. The values of k(0)/K(m) (s(-1). mM(-1)) of the BT-mutant for maltose and isomaltose were 69.0 and 15.4, respectively. We conclude that the Val/Ala200 and Pro/Asn258 residues in the alpha-glucosidases may be largely responsible for substrate recognition, although the regions I and IV also exert a slight influence. Additionally, BT V200A and V200A/P258N possessed high hydrolase activity towards sucrose.

Characterization of the export of bulk poly(A)+ mRNA in Saccharomyces cerevisiae during the wine-making process.

Ethanol stress affects the nuclear export of mRNA similarly to heat shock in Saccharomyces cerevisiae. However, we have little information about mRNA transport in actual alcoholic fermentation. Here we characterized the transport of mRNA during wine making and found that bulk poly(A)+ mRNA accumulated in the nucleus as fermentation progressed.

Characterization of Rat8 localization and mRNA export in Saccharomyces cerevisiae during the brewing of Japanese sake.

Ethanol affects the nuclear export of mRNA in a similar way to heat shock in Saccharomyces cerevisiae. We recently reported that the nuclear accumulation of Rat8 caused by ethanol stress correlates well with blocking of the export of bulk poly(A)(+) mRNA. Here, we characterize the localization of Rat8 and bulk poly(A)(+) mRNA in sake (Japanese rice wine) yeast during the brewing of sake. In wine must and synthetic dextrose medium, sake yeast showed the same responses to ethanol regarding changes in the localization of Rat8 as wine yeast and a laboratory strain: i.e., cells began the nuclear accumulation of Rat8 at an ethanol concentration of 6% and completed it at 9%. In contrast, during the sake-brewing process, sake yeast showed unique phenomena: i.e., cells did not start the nuclear accumulation of Rat8 until the ethanol concentration of the sake mash reached around 12% and they showed a normal localization of Rat8 around the nuclear envelope at the late stage of fermentation. These results provide new information about the transport of mRNA in yeast cells during actual alcoholic fermentation.

Molecular motors and mechanisms of directional transport in neurons.

Intracellular transport is fundamental for neuronal morphogenesis, function and survival. Many proteins are selectively transported to either axons or dendrites. In addition, some specific mRNAs are transported to dendrites for local translation. Proteins of the kinesin superfamily participate in selective transport by using adaptor or scaffolding proteins to recognize and bind cargoes. The molecular components of RNA-transporting granules have been identified, and it is becoming clear how cargoes are directed to axons and dendrites by kinesin superfamily proteins. Here we discuss the molecular mechanisms of directional axonal and dendritic transport with specific emphasis on the role of motor proteins and their mechanisms of cargo recognition.

Kinesin superfamily proteins and their various functions and dynamics.

Kinesin superfamily proteins (KIFs) are motor proteins that transport membranous organelles and macromolecules fundamental for cellular functions along microtubules. Their roles in transport in axons and dendrites have been studied extensively, but KIFs are also used in intracellular transport in general. Recent findings have revealed that in many cases, the specific interaction of cargoes and motors is mediated via adaptor/scaffolding proteins. Cargoes are sorted to precise destinations, such as axons or dendrites. KIFs also participate in polarized transport in epithelial cells as shown in the apical transport of annexin XIIIb-containing vesicles by KIFC3. KIFs play important roles in higher order neuronal activity; transgenic mice overexpressing KIF17, which transports N-methyl-d-asp (NMDA) receptors to dendrites, show enhanced memory and learning. KIFs also play significant roles in neuronal development and brain wiring: KIF2A suppresses elongation of axon collaterals by its unique microtubule-depolymerizing activity. X-ray crystallography has revealed the structural uniqueness of KIF2 underlying the microtubule-depolymerizing activity. In addition, single molecule biophysics and optical trapping have shown that the motility of monomeric KIF1A is caused by biased Brownian movement, and X-ray crystallography has shown how the conformational changes occur for KIF1A to move during ATP hydrolysis. These multiple approaches in analyzing KIF functions will illuminate many basic mechanisms underlying intracellular events and will be a very promising and fruitful area for future studies.

Molecular motors in neuronal development, intracellular transport and diseases.

Molecular motors such as kinesin superfamily proteins (KIFs), dynein superfamily proteins and myosin superfamily proteins have diverse and fundamental roles in many cellular processes, including neuronal development and the pathogenesis of neuronal diseases. During neuronal development, KIFs take significant roles in the regulation of axon-collateral branch extension, which is essential for brain wiring. Cytoplasmic dynein together with LIS1 takes pivotal roles in neocortical layer formation. In axons, anterograde transport is mediated by KIFs, whereas retrograde transport is mediated mainly by cytoplasmic dynein, and dysfunction of motors results in neurodegenerative diseases. In dendrites, the transport of NMDA and AMPA receptors is mediated by KIFs, and the motor has been shown to play a significant part in establishing learning and memory.

Stress response in yeast mRNA export factor: reversible changes in Rat8p localization are caused by ethanol stress but not heat shock.

Ethanol stress (10% v/v) causes selective mRNA export in Saccharomyces cerevisiae in a similar manner to heat shock (42 degrees C). Bulk poly(A)(+) mRNA accumulates in the nucleus, whereas heat shock protein mRNA is exported under such conditions. Here we investigated the effects of stress on mRNA export factors. In cells treated with ethanol stress, the DEAD box protein Rat8p showed a rapid and reversible change in its localization, accumulating in the nucleus. This change correlated closely with the blocking of bulk poly(A)(+) mRNA export caused by ethanol stress. We also found that the nuclear accumulation of Rat8p is caused by a defect in the Xpo1p/Crm1p exportin. Intriguingly, the localization of Rat8p did not change in heat shocked cells, suggesting that the mechanisms blocking bulk poly(A)(+) mRNA export differ for heat shock and ethanol stress. These results suggest that changes in the localization of Rat8p contribute to the selective export of mRNA in ethanol stressed cells, and also indicate differences in mRNA export between the heat shock response and ethanol stress response.

Gle2p is essential to induce adaptation of the export of bulk poly(A)+ mRNA to heat shock in Saccharomyces cerevisiae.

The export of bulk poly(A)(+) mRNA is blocked under heat-shocked (42 degrees C) conditions in Saccharomyces cerevisiae. We found that an mRNA export factor Gle2p rapidly dissociated from the nuclear envelope and diffused into the cytoplasm at 42 degrees C. However, in exponential phase cells pretreated with mild heat stress (37 degrees C for 1 h), Gle2p did not dissociate at 42 degrees C, and the export of bulk poly(A)(+) mRNA continued. Cells in stationary phase also continued with the export of bulk poly(A)(+) mRNA at 42 degrees C without the dissociation of Gle2p from the nuclear envelope. The dissociation of Gle2p was caused by increased membrane fluidity and correlated closely with blocking of the export of bulk poly(A)(+) mRNA. Furthermore, the mutants gle2Delta and rip1Delta could not induce such an adaptation of the export of bulk poly(A)(+) mRNA to heat shock. Our findings indicate that Gle2p plays a crucial role in mRNA export especially under heat-shocked conditions. Our findings also indicate that the nuclear pore complexes that Gle2p constitutes need to be stabilized for the adaptation and that the increased membrane integrity caused by treatment with mild heat stress or by survival in stationary phase is likely to contribute to the stabilization of the association between Gle2p and the nuclear pore complexes.

Biochemical and molecular characterization of diseases linked to motor proteins.

Recent studies have revealed that kinesin, dynein and myosin each form large superfamilies and participate in many different intracellular transport systems. Importantly, these motor proteins play significant roles in the pathogenesis of a variety of diseases. Studies using knockout mice for kinesin KIF1B have led to the identification of the cause of a human hereditary neuropathy, Charcot-Marie-Tooth disease type 2A. The function of members of the dynein superfamily whose existence has previously only been confirmed through genome databases, has been revealed by studies of immotile cilia syndrome. Unconventional myosins have been shown to function in the inner-ear cells by examination of hereditary human hearing impairment and studies using mouse models. In addition, some diseases are caused by mutations, not in the motor itself, but in the proteins associated with the motor proteins. Here, we discuss the relationship of these motor proteins and how they contribute to disease in molecular terms.