Prognostic impact of residual lateral lymph node metastasis after neoadjuvant (chemo)radiotherapy in patients with advanced low rectal cancer.

Background: There is a lack of large studies focusing on the prognostic significance of lateral lymph node (LLN) metastasis following LLN dissection (LLND) in rectal cancer. The aim of this study was to evaluate the prognostic impact of LLN metastases on survival of patients with advanced low rectal cancer. Methods: Consecutive patients with locally advanced, but not metastatic, extraperitoneal rectal cancer treated with neoadjuvant (chemo)radiotherapy plus total mesorectal excision between 2004 and 2015 were included in the study. LLND was performed when pretreatment imaging documented enlarged LLNs (7 mm or greater in size). Localization of nodal metastases and long-term outcomes were analysed. Kaplan-Meier analysis was used to compare the survival of patients with ypN0 disease with that of patients with mesorectal ypN+/LLN- status and patients with positive LLNs. The Cox proportional hazards model was used to evaluate predictors of disease-free survival (DFS) and local recurrence. Results: A total of 613 patients were included in the study; LLND was performed in 212 patients (34·6 per cent) and 57 (9·3 per cent) had LLN metastasis. Patients with LLN metastasis had improved DFS and local recurrence cumulative incidence rates compared with patients with mesorectal ypN2+/LLN- disease (DFS: P = 0·014; local recurrence: P = 0·006). Although the DFS rate of patients with LLN metastasis was worse than that of patients with ypN0 disease (P < 0·001), the cumulative incidence of local recurrence was similar (P = 0·491). In multivariable analysis, residual LLN metastasis was not an independent predictor of worse DFS or local recurrence. Conclusion: LLN metastasis is not an independent predictor of local recurrence or survival. Survival of patients presenting with LLN metastasis after (chemo)radiotherapy was intermediate between that of patients with ypN0 status and those with mesorectal ypN2 positivity.

Antecedentes: No existen en la literatura grandes estudios dirigidos a investigar la importancia pronóstica de las metástasis en los ganglios linfáticos laterales (lateral lymph nodes, LLN) después de la disección de los mismos (LLN dissection, LLND) en pacientes con cáncer de recto. El objetivo de este estudio fue evaluar el impacto pronóstico de las metástasis en los LLN sobre la supervivencia de los pacientes con cáncer de recto. Métodos: Se analizaron 613 pacientes consecutivos con cáncer de recto localmente avanzado extraperitoneal y no metastásico tratados con (quimio)radioterapia neoadyuvante seguida de resección total del mesorrecto (total mesorectal excision, TME) entre 2004 y 2015. Se realizó una LLND cuando el estudio mediante pruebas de imagen previo el tratamiento mostró LLN aumentados de tamaño ≥ 7 mm. Se analizó la localización de las metástasis ganglionares y los resultados a largo plazo. El análisis de supervivencia se realizó mediante el método de Kaplan­Meier para comparar las supervivencias de los pacientes ypN0 frente a los pacientes ypN con positividad mesorrectal/LLN negativos y frente a los pacientes LLN positivos. Se utilizó el modelo de riesgo proporcional de Cox para evaluar los factores predictivos de supervivencia libre de enfermedad y de recidiva local. Resultados: Se realizó una LLND en 212 (34,6%) pacientes, y 57 (9,3%) pacientes presentaban metástasis en los LLN. Los pacientes con metástasis en los LLN presentaron mejores curvas de incidencia acumulada de recidiva local y de supervivencia libre de enfermedad en comparación con los pacientes con ganglios mesorrectales ypN2 positivos/LLN negativos (respectivamente, P = 0,0135 y P = 0,0060). Aunque la curva de la supervivencia libre de enfermedad de los pacientes con metástasis en los LLN fue peor que la de los pacientes ypN0 (P < 0,0001), la incidencia acumulada de recidiva local fue similar (P = 0,4905). En el análisis multivariable, la metástasis residual en los LLN no fue un factor predictivo independiente de peor supervivencia libre de enfermedad ni de recidiva local. Conclusión: Las metástasis en los LLN no es un factor predictivo independiente de recidiva local o supervivencia. Los pacientes que presentaron metástasis en los LLN después de (quimio)radioterapia mostraron características de supervivencia intermedias entre ypN0 y pacientes con ganglios mesorrectales ypN2 positivos.

Comparison of preoperative whole-body positron emission tomography with MDCT in patients with primary colorectal cancer.

OBJECTIVE: Preoperative use of emission tomography with(18)F-fluorodeoxyglucose (FDG-PET) in patients with primary colorectal cancer remains controversial. This study evaluated the additional value of FDG-PET in comparison with routine multidetector row computed tomography (MDCT) in patients with primary colorectal cancer. METHOD: Retrospective analysis was performed in 65 patients with colorectal cancer who underwent whole-body FDG-PET. Results of FDG-PET were compared with routine preoperative evaluation by MDCT regarding detection of primary tumour, lymph node involvement and distant metastases. All images were evaluated before surgery. RESULTS: Tumour detection rate was 100% (63/63) for MDCT and 98% (62/63) for FDG-PET. Lymph node involvement was pathologically confirmed in 35 patients. MDCT and FDG-PET displayed sensitivities of 89% (31/35; 95% CI: 73-97%) and 43% (15/35; 95% CI: 26-61%) and specificities of 52% (11/21; 95% CI: 30-74%) and 95% (20/21; 95% CI: 76-100%), respectively. Liver metastases were present in 22 patients. MDCT and FDG-PET showed accuracies of 98% (64/65; 95% CI: 92-100%) and 97% (63/65; 95% CI: 89-100%), respectively. FDG-PET detected additional extrahepatic metastatic lesions and affected treatment plan compared with MDCT in 10 patients. CONCLUSION: Preoperative FDG-PET is not superior to MDCT for detection of primary tumour, lymph node involvement or liver metastases, but may have potential clinical value in patients with advanced colorectal cancer by detecting extrahepatic distant metastases.

Mouse homologue of coq7/clk-1, longevity gene in Caenorhabditis elegans, is essential for coenzyme Q synthesis, maintenance of mitochondrial integrity, and neurogenesis.

coq7/clk-1 was isolated from a long-lived mutant of Caenorhabditis elegans, which showed sluggish behavior and an extended life span. Mouse coq7 is homologous to Saccharomyces cerevisiae coq7/cat5 that is required for biosynthesis of coenzyme Q (CoQ), an essential cofactor in mitochondrial respiration. Here we generated COQ7-deficient mice to investigate the biological role of COQ7 in mammals. COQ7-deficient mouse embryos failed to survive beyond embryonic day 10.5, exhibiting small-sized body and delayed embryogenesis. Morphological studies showed that COQ7-deficient neuroepithelial cells failed to show the radial arrangement in the developing cerebral wall, aborting neurogenesis at E10.5. Electron microscopic analysis further showed the enlarged mitochondria with vesicular cristae and enlarged lysosomes filled with disrupted membranes, which is consistent with mitochondriopathy. Biochemical analysis demonstrated that COQ7-deficient embryos failed to synthesize CoQ(9), but instead yielded demethoxyubiquinone 9 (DMQ(9)). Cultured embryonic cells from COQ7-deficient mice were rescued by adding bovine fetal serum in vitro, but exhibited slowed cell proliferation, which resembled to the phenotype of clk-1 with delayed cell divisions. The result implied the essential role of coq7 in CoQ synthesis, maintenance of mitochondrial integrity, and neurogenesis in mice.

Mouse coq7/clk-1 orthologue rescued slowed rhythmic behavior and extended life span of clk-1 longevity mutant in Caenorhabditis elegans.

The coq7/clk-1 gene was isolated from the long-lived mutant of Caenorhabditis elegans and was suggested to play a regulatory role in biological rhythm and longevity. The mouse COQ7 is homologous to Saccharomyces cerevisiae COQ7/CAT5 that is required for the biosynthesis of coenzyme Q (ubiquinone), an essential messenger in mitochondrial respiration. In the present study, we characterized the expression and processing of mouse COQ7. We found that COQ7 is highly expressed in tissues with high energy demand such as heart, muscle, liver, and kidney in mice. Biochemical analysis revealed that COQ7 is targeted to mitochondria where it is processed to mature form. Transgenic expression of mouse coq7 completely rescued the slowed rhythmic behaviors of clk-1 such as defecation. In life-span analysis, transgenic expression reverted the extended life span of clk-1 to the comparable level with wild-type control. These data strongly suggested that coq7 plays a pivotal role in the regulation of biological rhythms and the determination of life span in mammalian species.

Aberrant synaptic transmission in the hippocampal CA3 region and cognitive deterioration in protein-repair enzyme-deficient mice.

L-aspartate is the amino-acid residue most susceptible to spontaneous isomerization. This denaturation causes an alteration in the biological activity of the protein and is regarded as an aging process of the protein. Protein L-isoaspartyl methyltransferase (PIMT) repairs this post-translational modification and thus is implicated in retarding the aging process of proteins. PIMT is highly expressed in the brain, and its deficiency results in progressive epilepsy after 4 weeks of age, with a fatal seizure in mice. Here we report the pathophysiological role of this repair system in the hippocampal slice of PIMT-deficient mice. The hippocampal mossy fiber-CA3 synapses of PIMT-deficient mice showed hyperexcitation that was repressed by a gamma-aminobutyric acid (GABA)A receptor agonist muscimol. In addition, the mossy fiber-CA3 synapses failed to show long-term potentiation or paired-pulse facilitation. No abnormality, however, was observed in Schaffer collateral-CA1 synapses or in perforant path-dentate gyrus synapses. Electron microscopic study revealed aberrant distribution of synaptic vesicles in the mossy fiber terminals and vacuolar degeneration at the axon hillock of dentate granule cells in PIMT-deficient mice. Furthermore, the PIMT-deficient mice showed impaired spatial memory in Morris water maze test and exhibited fewer anxiety-related behaviors in the elevated-plus test. These results suggest that the mossy fiber-CA3 system is vulnerable to aspartate isomerization and that the PIMT-mediated repair system is essential for maintenance of normal functions of the hippocampus.

SPK-1, a C. elegans SR protein kinase homologue, is essential for embryogenesis and required for germline development.

SR-protein kinases (SRPKs) and their substrates, serine/arginine-rich pre-mRNA splicing factors, are key components of splicing machinery and are well conserved across phyla. Despite extensive biochemical investigation, the physiological functions of SRPKs remain unclear. In the present study, cDNAs for SPK-1, a C. elegans SRPK homologue, and CeSF2, an SPK-1 substrate, were cloned. SPK-1 binds directly to and phosphorylates the RS domain of CeSF2 in vitro. Both spk-1 and CeSF2 are predominantly expressed in germlines. RNA interference (RNAi) experiments revealed that spk-1 and CeSF2 play an essential role at the embryonic stage of C. elegans. Furthermore, RNAi studies demonstrated that spk-1 is required for germline development in C. elegans. We provide evidence that RNAi, achieved by the soaking of L1 larvae, is beneficial in the study of gene function in post-embryonic germline development.

Mouse ULK2, a novel member of the UNC-51-like protein kinases: unique features of functional domains.

The UNC-51 serine/threonine kinase of C. elegans plays an essential role in axonal elongation, and unc-51 mutants exhibit uncoordinated movements. We have previously identified mouse and human cDNAs encoding UNC-51-like kinase (ULK1). Here we report the identification and characterization of the second murine member of this kinase family, ULK2. Mouse ULK2 cDNA encodes a putative polypeptide of 1033 aa which has an overall 52% and 33% amino acid identity to ULK1 and UNC-51, respectively. ULKs and UNC-51 share a typical domain structure of an amino-terminal kinase domain, a central proline/serine rich (PS) domain, and a carboxy-terminal (C) domain. Northern blot analysis showed that ULK2 mRNA is widely expressed in adult tissues. In situ hybridization analysis indicated that ULK2 mRNA is ubiquitously localized in premature as well as mature neurons in developing nervous system. ULK2 gene was mapped to mouse chromosome 11B1.3 and rat chromosome 10q23 by FISH. HA-tagged ULK2 expressed in COS7 cells had an apparent molecular size of approximately 150 kDa and was autophosphorylated in vitro. Truncation mutants suggested that the autophosphorylation occurs in the PS domain. Although expression of ULK2 failed to rescue unc-51 mutant of C. elegans, a series of ULK2/UNC-51 chimeric kinases revealed that function of the kinase and PS domains are conserved among species, while the C domain acts in a species-specific manner. These results suggest that ULK2 is involved in a previously uncharacterized signaling pathway in mammalian cells.

Orthologues of the Caenorhabditis elegans longevity gene clk-1 in mouse and human.

The clk-1 gene was isolated from the long-lived mutant of Caenorhabditis elegans and was suggested to play a biological role in longevity (Ewbank et al., 1997, Science 275: 980-983). The primary structure of CLK-1 showed a significant homology to Saccharomyces cerevisiae Coq7p/Cat5p, which is required for the biosynthesis of ubiquinone and the derepression of gluconeogenic genes. In the present study, we isolated and characterized human and mouse orthologues of the COQ7/CLK-1 gene. Sequence analysis of both the human and the mouse COQ7 cDNAs showed an open reading frame composed of 217 amino acids with calculated molecular mass of 24,309 and 24,044 Da, respectively. Homology search revealed that human COQ7 showed 85% identity to mouse COQ7, 89% identity to rat COQ7, 53% identity to C. elegans CLK-1, and 37% identity to S. cerevisiae Coq7p/Cat5p. Zoo blot analysis implied that the COQ7 gene was well conserved among mammal, bird, and reptile genomes. Tissue blot analysis showed that human COQ7 is dominantly transcribed in heart and skeletal muscle. Genomic analyses revealed that the human COQ7 gene is composed of six exons spanning 11 kb of human genome as a single-copy gene. Radiation hybrid mapping assigned the COQ7 gene to human chromosome 16p12.3-p13.11.

A mouse serine/threonine kinase homologous to C. elegans UNC51 functions in parallel fiber formation of cerebellar granule neurons.

The formation of the cerebellar circuitry depends on the outgrowth of connections between the two principal classes of neurons, granule neurons and Purkinje neurons. To identify genes that function in axon outgrowth, we have isolated a mouse homolog of C. elegans UNC51, which is required for axon formation, and tested its function in cerebellar granule neurons. Murine Unc51.1 encodes a novel serine/threonine kinase and is expressed in granule cells in the cerebellar cortex. Retroviral infection of immature granule cells with a dominant negative Unc51.1 results in inhibition of neurite outgrowth in vitro and in vivo. Moreover, infected neurons fail to express TAG-1 or neuron-specific beta-tubulin, suggesting that development is arrested prior to this initial step of differentiation. Thus, Unc51.1 signals the program of gene expression leading to the formation of granule cell axons.

Human ULK1, a novel serine/threonine kinase related to UNC-51 kinase of Caenorhabditis elegans: cDNA cloning, expression, and chromosomal assignment.

The unc-51 gene, isolated from mutants of Caenorhabditis elegans exhibiting abnormal axonal extension and growth, encodes a novel serine/threonine kinase (K. Ogura, et al., 1994, Genes Dev. 8: 2389-2400). Here we report the molecular cloning and characterization of the human homologue of UNC-51, designated ULK1, for UNC-51 (C. elegans)-like kinase 1. Sequence analysis of the human ULK1 cDNA showed that an open reading frame is composed of 1050 amino acids with a calculated MW of 112.6 kDa and a pI of 8.80. Homology search analysis showed that ULK1 has 41% overall similarity to UNC-51 and 29% similarity to Apg1p of Saccharomyces cerevisiae. Phylogenetic analysis of ULK1, UNC-51, and Apglp suggested that they constitute a novel subfamily of serine/threonine kinases. Southern blot analyses suggested that the ULK1 gene spans 30-40 kb in the human genome as a single-copy gene. Zoo blot analysis indicated that ULK1 kinase is conserved among vertebrates including mammals, birds, reptiles, amphibians, and fish. Northern blot analysis revealed that ULK1 is ubiquitously expressed in adult human tissues such as skeletal muscle, heart, pancreas, brain, placenta, liver, kidney, and lung, whereas UNC-51 is specifically detected in the nervous system of C. elegans. Both FISH and RH mapping confirmed the regional localization of ULK1 to human chromosome 12q24.3.

Identification of mouse ULK1, a novel protein kinase structurally related to C. elegans UNC-51.

A novel protein kinase related to the C. elegans serine/threonine kinase UNC-51 was cloned from mouse. The UNC-51-Like Kinase (ULK)1 is encoded by a cDNA of 1051 amino acids with calculated MW of 113 kDa. Comparison of the ULK1 and UNC-51 shows the highest conservation in the amino-terminal kinase domain, which is followed by a proline/serine-rich (PS) domain and a conserved carboxyl-terminal (C) domain. ULK1 mRNA is expressed in various tissues, and is mapped to mouse chromosome 5F and rat chromosome 12q16.3, by fluorescent in situ hybridization. HA-tagged ULK1 is expressed as a protein of approximately 150 kDa in COS7 cells and is auto-phosphorylated in vitro in its PS domain. We propose that ULK1, UNC-51 and a yeast protein kinase Apg1p comprise a novel subfamily of protein kinase, which is structurally conserved among eukaryotes.

Deficiency in protein L-isoaspartyl methyltransferase results in a fatal progressive epilepsy.

Protein L-isoaspartyl methyltransferase (PIMT) is suggested to play a role in the repair of aged protein spontaneously incorporated with isoaspartyl residues. We generated PIMT-deficient mice by targeted disruption of the PIMT gene to elucidate the biological role of the gene in vivo. PIMT-deficient mice died from progressive epileptic seizures with grand mal and myoclonus between 4 and 12 weeks of age. An anticonvulsive drug, dipropylacetic acid (DPA), improved their survival but failed to cure the fatal outcome. L-Isoaspartatate, the putative substrate for PIMT, was increased ninefold in the brains of PIMT-deficient mice. The brains of PIMT-deficient mice started to enlarge after 4 weeks of age when the apical dendrites of pyramidal neurons in cerebral cortices showed aberrant arborizations with disorganized microtubules. We conclude that methylation of modified proteins with isoaspartyl residues is essential for the maintenance of a mature CNS and that a deficiency in PIMT results in fatal progressive epilepsy in mice.

cDNA cloning, expression, subcellular localization, and chromosomal assignment of mammalian aurora homologues, aurora-related kinase (ARK) 1 and 2.

Chromosomal segregation during mitosis as well as meiosis is considered to be regulated by multiple kinases, but the precise mechanism remains largely unknown. A mutation in Drosophila, designated aurora, was identified as a responsible gene for a chromosomal segregation defect and encodes a putative serine-threonine kinase. Here we have identified mammalian aurora homologues, designated aurora-related kinase (ARK) 1 and ARK2. Kinase domains of murine ARK1 and ARK2 showed 61 and 62% identity, respectively, to that of aurora at the amino acid levels, respectively. Cell cycle analysis revealed that the expression of ARK1 was correlated with G2/M phase, while ARK2 was expressed during S and G2/M phases. Immunofluorescence analysis demonstrated that ARK2 was mainly localized to the midbody, while ARK1 has been reported to be localized to the spindle pole during mitosis. Collectively, these results suggest that these two kinases may have distinct roles with different expression timing and subcellular localization during the cell cycle progression. Interspecific backcross mapping revealed that Ark1 is located in a distal region of mouse chromosome 2, while Ark2 is located in a central region of mouse chromosome 11.

Induction of protein tyrosine phosphatase epsilon transcripts during NGF-induced neuronal differentiation of PC12D cells and during the development of the cerebellum.

We have investigated a possible role played by protein tyrosine phosphatase epsilon (PTPepsilon), which was recently cloned and predominantly expressed in brain, in neural differentiation and function. During neuronal cell differentiation of PC12D cells triggered by NGF or FGF, PTPepsilon transcripts were transiently induced at a time between the appearance of transcripts for immediate-early genes and for neuronal cell-specific markers. PTPepsilon was the only PTPase whose transcripts were induced during PC12D cell differentiation among over two dozen PTPase transcripts so far examined. Moreover, in situ hybridization revealed that PTPepsilon transcripts were detected in the neural tube of day 12 postcoitum embryo, and in the nervous system including brain, spinal cord, and ganglions in a ubiquitous manner in late gestational stages. In 4-day-old neonatal mice, the transcripts were widely distributed in the central nervous system where the strongest expression was detected in the hippocampus, cerebral cortex, and olfactory bulb. Interestingly, in day 7 and 16 neonatal brains, the strongest PTPepsilon gene expression was localized in the granular cells of cerebellum, which might indicate that PTPepsilon is involved in the differentiation of the granular cells. The biological significance of PTPepsilon in neuronal differentiation and brain functions is discussed.

Glutamate dehydrogenase mRNA is immediately induced after phencyclidine treatment in the rat brain.

To clarify the molecular mechanism of phencyclidine (PCP)-induced schizophreniform psychosis in humans and of behavioral abnormalities in experimental animals, we used differential screening of a cDNA library from the cerebral cortex of rats treated with PCP. We identified a PCP-induced cDNA clone as the gene encoding glutamate dehydrogenase (GDH), an enzyme central to glutamate metabolism. GDH mRNA levels significantly increased as early as 15 min following PCP administration in both the cerebral cortex and the cerebellum. This effect was observed even in the presence of a protein synthesis inhibitor, cycloheximide. In contrast to a transient increase in c-fos expression, the elevation of GDH mRNA levels lasted up to 8 days after a single PCP injection. These results suggest that GDH mRNA induction may be involved in the pathology of PCP-induced psychosis, and that GDH may be one of the candidate genes that are vulnerable in subjects with schizophrenia.

Identification of a receptor-type protein tyrosine phosphatase expressed in postmitotic maturing neurons: its structure and expression in the central nervous system.

We have isolated a rat cDNA encoding a receptor-type protein-tyrosine-phosphatase (RTP) expressed in brain and kidney (RPTP-BK) and characterized its expression in the developing central nervous system. RPTP-BK has seven fibronectin type III-like repeats in the extracellular region and a unique catalytic phosphatase domain in the cytoplasmic region. Bacterial expression of its phosphatase domain showed that the dephosphorylation of phosphotyrosine residues was mediated by the cytoplasmic catalytic domain. Sequence comparison revealed that RPTP-BK is homologous with GLEPP1, a rabbit PTP expressed in renal glomerular epithelia, and has the same phosphatase domain as murine PTPphi expressed in macrophages. RPTP-BK has also significant homology with Drosophila DPTP10D in the phosphatase domain, whose expression is localized exclusively in growth cones of the embryonal brains. The gene for RPTP-BK is well conserved among other species, and the expression in the brain but not in the kidney is developmentally regulated during the neonatal stage. Hybridization in situ showed that RPTP-BK is highly expressed in the postmitotic maturing neurons of the olfactory bulb, developing neocortex, hippocampus and thalamus. Because the expression of RPTP-BK in the developing neocortex is correlated with the stage of axonogenesis in cortical neurons, RPTP-BK might be crucial in neural cell development of the mammalian central nervous system.