Makoto Kawaguchi

Comparison of lung cancer cell lines representing four histopathological subtypes with gene expression profiling using quantitative real-time PCR

BackgroundLung cancers are the most common type of human malignancy and are intractable. Lung cancers are generally classified into four histopathological subtypes: adenocarcinoma (AD), squamous cell carcinoma (SQ), large cell carcinoma (LC), and small cell carcinoma (SC). Molecular biological characterization of these subtypes has been performed mainly using DNA microarrays. In this study, we compared the gene expression profiles of these four subtypes using twelve human lung cancer cell lines and the more reliable quantitative real-time PCR (qPCR).ResultsWe selected 100 genes from public DNA microarray data and examined them by DNA microarray analysis in eight test cell lines (A549, ABC-1, EBC-1, LK-2, LU65, LU99, STC 1, RERF-LC-MA) and a normal control lung cell line (MRC-9). From this, we extracted 19 candidate genes. We quantified the expression of the 19 genes and a housekeeping gene, GAPDH, with qPCR, using the same eight cell lines plus four additional validation lung cancer cell lines (RERF-LC-MS, LC-1/sq, 86-2, and MS-1-L). Finally, we characterized the four subtypes of lung cancer cell lines using principal component analysis (PCA) of gene expression profiling for 12 of the 19 genes (AMY2A, CDH1, FOXG1, IGSF3, ISL1, MALL, PLAU, RAB25, S100P, SLCO4A1, STMN1, and TGM2). The combined PCA and gene pathway analyses suggested that these genes were related to cell adhesion, growth, and invasion. S100P in AD cells and CDH1 in AD and SQ cells were identified as candidate markers of these lung cancer subtypes based on their upregulation and the results of PCA analysis. Immunohistochemistry for S100P and RAB25 was closely correlated to gene expression.ConclusionsThese results show that the four subtypes, represented by 12 lung cancer cell lines, were well characterized using qPCR and PCA for the 12 genes examined. Certain genes, in particular S100P and CDH1, may be especially important for distinguishing the different subtypes. Our results confirm that qPCR and PCA analysis provide a useful tool for characterizing cancer cell subtypes, and we discuss the possible clinical applications of this approach.

Essential developmental, genomic stability, and tumour suppressor functions of the mouse orthologue of hSSB1/NABP2.

Single-stranded DNA binding proteins (SSBs) regulate multiple DNA transactions, including replication, transcription, and repair. We recently identified SSB1 as a novel protein critical for the initiation of ATM signaling and DNA double-strand break repair by homologous recombination. Here we report that germline Ssb1−/− embryos die at birth from respiratory failure due to severe rib cage malformation and impaired alveolar development, coupled with additional skeletal defects. Unexpectedly, Ssb1 −/− fibroblasts did not exhibit defects in Atm signaling or γ-H2ax focus kinetics in response to ionizing radiation (IR), and B-cell specific deletion of Ssb1 did not affect class-switch recombination in vitro. However, conditional deletion of Ssb1 in adult mice led to increased cancer susceptibility with broad tumour spectrum, impaired male fertility with testicular degeneration, and increased radiosensitivity and IR–induced chromosome breaks in vivo. Collectively, these results demonstrate essential roles of Ssb1 in embryogenesis, spermatogenesis, and genome stability in vivo.

Prediction of pathological and oncological outcomes based on extended prostate biopsy results in patients with prostate cancer receiving radical prostatectomy: a single institution study

BackgroundThe prediction of pathological outcomes prior to surgery remains a challenging problem for the appropriate surgical indication of prostate cancer. This study was performed to identify preoperative values predictive of pathological and oncological outcomes based on standardized extended prostate biopsies with core histological results diagrammed/mapped in patients receiving radical prostatectomy for prostate cancer clinically diagnosed as localized or locally advanced disease.MethodsIn 124 patients with clinically localized or locally advanced prostate cancer (cT1c–cT3a) without prior treatment, pathological outcomes on the surgical specimen including seminal vesicle involvement (SVI), positive surgical margin (PSM), and perineural invasion (PNI) were studied in comparison with clinical parameters based on the results of 14-core prostate biopsies comprising sextant, laterally-directed sextant, and bilateral transition zone (TZ) sampling.ResultsConcerning the association of pathological outcomes with oncological outcomes, patients with PSM and PNI on surgical specimens had poorer biochemical-progression-free survival than those without PSM (logrank pu2009=u20090.002) and PNI (pu2009=u20090.003); it was also poorer concerning SVI, although the difference was not significant (pu2009=u20090.120). Concerning the impact of clinical parameters on these pathological outcomes, positive TZ and multiple positive biopsy cores in the prostatic middle were independent values predictive of SVI with multivariate analyses (pu2009=u20090.020 and pu2009=u20090.025, respectively); both positive TZ and multiple positive prostatic middle biopsies were associated with larger tumor volume (pu2009<u20090.001 in both). The percentage of positive biopsy cores (%positive cores) and biopsy Gleason score were independent values predictive of PSM (pu2009=u20090.001) and PNI (pu2009=u20090.001), respectively. Multiple positive cores in the prostatic base were associated with proximal/bladder-side PSM (pu2009<u20090.001), and also linked to poorer biochemical-progression-free survival (pu2009=u20090.004). Clinical T stage had no association with these pathological outcomes.Conclusions%positive cores and Gleason score in extended biopsies were independent values predictive of PSM and PNI in prostate cancer clinically diagnosed as localized or locally advanced disease, respectively, which were associated with poorer oncological outcomes. When diagramming biopsy-core results, extended biopsy may provide additional information for predicting oncological and pathological outcomes including SVI in patients clinically diagnosed as having localized or locally advanced disease.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/8790262771042628

Low‐grade renal epithelial tumor originating from the distal nephron

There are few published reports of low-grade renal epithelial tumor originating from the distal nephron. However, it should not be disregarded clinically, because the actual number of patients with such tumors may be higher than expected. We investigated the immunohistochemical profile of a histologically distinct subtype of such a tumor in detail, in addition to the clinical course and imaging studies. The present study demonstrated that both glandular and spindle cell components of this tumor have a persistent characteristic of an epithelial tumor arising from the distal tubule or collecting duct. This tumor is a benign complex neoplasm that can be treated successfully with radical surgery. Beta-catenin and E-cadherin are suggested to play a crucial role in tumorigenesis and the biphasic arrangement of this neoplasm, concerning the expression of epithelial membrane antigen and carbohydrate antigen 19-9. We suggest that the term distal nephron epithelioma is appropriate for classifying such rare but clinicopathologically distinct tumors.In the February 2004 issue of International Journal of Urology , Hara et al . reported a case of a low-grade renal epithelial tumor originating from the distal nephron. 1 I read their article with great interest. Regarding this neoplasm, two large studies have been reported in 2002. 2,3 Rakozy et al . reported multiple losses of chromosomes 1, 4, 6, 8, 9, 13, 14, 15 and 22 in low-grade tubularmucinous renal neoplasm (LGTMRN) by using comparative genomic hybridization. 3 Subsequently, this neoplasm has been categorized as mucinous tubular and spindle cell carcinoma (MTSCC), according to the World Health Organization classification of tumors. 4

Median raphe cyst in the scrotum, mimicking a serous borderline tumor, associated with cryptorchidism after orchiopecxy

Abstractu2002 Median raphe cyst (MRC) is a benign lesion occurring predominantly in the ventral surface of the penises of young men and is an embryological developmental anomaly of the male genitalia. Serous borderline tumors (SBT) are found most frequently in the female ovary and only several cases with SBT of the male genitalia have been reported. We describe a case of MRC with features of SBT, which appeared in the scrotum of a 9‐year‐old boy after orchiopexy and was associated with surgery for cryptorchidism. The cyst arose on the right testicular tunica and consisted of cystic components with intracystic papillae lined by stratified epithelial cells, some of which showed mild cytological atypia and sporadic mitosis. These epithelial cells expressed CAu2003125, CAu200319‐9, carcinoembryonic antigen, estrogen receptor and progesterone receptor. Although no cases of MRC with characteristics of SBT in association with the rete testis has been described, the current report gives additional information for follow‐up of cryptorchidism.

Leiomyoma originating from the extrapleural tissue of the chest wall

We describe a rare case of leiomyoma of the chest wall in a 55-year-old female. Computed tomography showed a well-circumscribed neoplasm with a diameter of 2 cm in the right chest wall. The tumor was excised with video-assisted thoracic surgery. Histopathology confirmed that the tumor was leiomyoma arising from the microvascular smooth muscle in the chest wall. We present the immunohistochemical profiles of the tumor in detail, critically reviewing the previously reported cases.

Parathyroid hormone‐related peptide‐producing non‐familial pheochromocytoma in a child

We experienced a case of parathyroid hormone‐related peptide (PTHrP)‐producing pheochromocytoma, which was found in a 12‐year‐old boy with hypercalcemia. The leading symptom was abdominal pain, and severe hypertension and tachycardia were noticed at the initial visit. His medical and familial histories were unremarkable. Laboratory examinations showed hypercalcemia (3.3u2003mmol/L of serum‐calcium). Computed tomography showed a heterogeneous mass measuring 5.0u2003cm in the right adrenal gland, which had abnormal uptake with 123‐I metaiodobenzylguanidine scintigraphy. Serum/urine catecholamines were highly elevated, and serum PTHrP also increased (1.4u2003pmol/L). The patient underwent laparoscopic right adrenalectomy. The tumor was histologically diagnosed as typical pheochromocytoma and the expression of PTHrP was confirmed with immunohistochemistry. The serum PTHrP level was normalized after surgery. He was free of disease postoperatively for 12u2003months. There has been no described pediatric patient with PTHrP‐producing pheochromocytoma. We showed evidence that the present tumor is a complex neoplasm involving various neuroendocrine activities with the dual‐lineage differentiation.

AT motif binding factor 1 (ATBF1) is highly phosphorylated in embryonic brain and protected from cleavage by calpain-1.

ATBF1 is a transcription factor that regulates genes responsible for repairing tissues and the protection of cells from oxidative stress. Therefore reduction of ATBF1 promotes susceptibility to varieties of human diseases including neurodegenerative diseases and malignant tumors. The instability of the protein was found to be an important background of diseases. Because ATBF1 is composed of a large 404-kDa protein, it can be easily targeted by proteinases. The protein instability should be a serious problem for the function in the cells and practically for our biochemical study of ATBF1. We have found that calpain-1 is a protease responsible for the degeneration of ATBF1. We observed distinct difference between embryo and adult brain derived ATBF1 regarding the sensitivity to calpain-1. The comparative study showed that eight phosphorylated serine residues (Ser1600, Ser2634, Ser2795, Ser2804, Ser2900, Ser3431, Ser3613, Ser3697) in embryonic brain, but only one site (Ser2634) in adult brain. As long as these amino acids were phosphorylated, ATBF1 derived from embryonic mouse brain showed resistance to cleavage; however, treatment with calf intestine alkaline phosphatase sensitized ATBF1 to be digested by calpain-1. An inhibitor (FK506) against calcineurin, which is a serine/threonine specific phosphatase enhanced the resistance of ATBF1 against the digestion by calpain-1. Taken together, these results demonstrate that these phosphorylation sites on ATBF1 function as a defensive shield to calpain-1.

Prolonged activation of IL-5–producing ILC2 causes pulmonary arterial hypertrophy

IL-33 is one of the critical cytokines that activates group 2 innate lymphoid cells (ILC2s) and mediates allergic reactions. Accumulating evidence suggests that IL-33 is also involved in the pathogenesis of several chronic inflammatory diseases. Previously, we generated an IL-5 reporter mouse and revealed that lung IL-5-producing ILC2s played essential roles in regulating eosinophil biology. In this study, we evaluated the consequences of IL-33 administration over a long period, and we observed significant expansion of ILC2s and eosinophils surrounding pulmonary arteries. Unexpectedly, pulmonary arteries showed severe occlusive hypertrophy that was ameliorated in IL-5- or eosinophil-deficient mice, but not in Rag2-deficient mice. This indicates that IL-5-producing ILC2s and eosinophils play pivotal roles in pulmonary arterial hypertrophy. Administration of a clinically used vasodilator was effective in reducing IL-33-induced hypertrophy and repressed the expansion of ILC2s and eosinophils. Taken together, these observations demonstrate a previously unrecognized mechanism in the development of pulmonary arterial hypertrophy and the causative roles of ILC2 in the process.

A diagnostic marker for superficial urothelial bladder carcinoma: lack of nuclear ATBF1 (ZFHX3) by immunohistochemistry suggests malignant progression.

BackgroundPathological stage and grade have limited ability to predict the outcomes of superficial urothelial bladder carcinoma at initial transurethral resection (TUR). AT-motif binding factor 1 (ATBF1) is a tumor suppressive transcription factor that is normally localized to the nucleus but has been detected in the cytoplasm in several cancers. Here, we examined the diagnostic value of the intracellular localization of ATBF1 as a marker for the identification of high risk urothelial bladder carcinoma.MethodsSeven anti-ATBF1 antibodies were generated to cover the entire ATBF1 sequence. Four human influenza hemagglutinin-derived amino acid sequence-tagged expression vectors with truncated ATBF1 cDNA were constructed to map the functional domains of nuclear localization signals (NLSs) with the consensus sequence KR[X10-12]K. A total of 117 samples from initial TUR of human bladder carcinomas were analyzed. None of the patients had received chemotherapy or radiotherapy before pathological evaluation.ResultsATBF1 nuclear localization was regulated synergistically by three NLSs on ATBF1. The cytoplasmic fragments of ATBF1 lacked NLSs. Patients were divided into two groups according to positive nuclear staining of ATBF1, and significant differences in overall survival (Pu2009=u20090.021) and intravesical recurrence-free survival (Pu2009=u20090.013) were detected between ATBF1+ (nu2009=u2009110) and ATBF1− (nu2009=u20097) cases. Multivariate analysis revealed that ATBF1 staining was an independent prognostic factor for intravesical recurrence-free survival after adjusting for cellular grading and pathological staging (Pu2009=u20090.008).ConclusionsCleavage of ATBF1 leads to the cytoplasmic localization of ATBF1 fragments and downregulates nuclear ATBF1. Alterations in the subcellular localization of ATBF1 due to fragmentation of the protein are related to the malignant character of urothelial carcinoma. Pathological evaluation using anti-ATBF1 antibodies enabled the identification of highly malignant cases that had been overlooked at initial TUR. Nuclear localization of ATBF1 indicates better prognosis of urothelial carcinoma.