Floral and pollination characteristics of Eriocaulon heleocharioides, an extinct species in the wild, for evidence-based conservation management.

Generally, floral characteristics and pollination are important factors enhancing the quality and quantity of reproductive output for regeneration in plant conservation. However, lack of evidence-based management could decrease fitness under ex-situ conservation. We investigated the capitulum and pollination characteristics of Eriocaulon heleocharioides Satake (Eriocaulaceae), which is extinct in the wild, to develop an evidence-based conservation management plan incorporating previously ignored reproductive characteristics. To evaluate the functional characteristics of capitula, pollen-ovule ratio, and reproductive status (maximum pollination success/florivory damage) were investigated along six flowering sequences of capitulum. To evaluate the effect of plant density on pollen transfer, high- and low-density plots were established. Total deposited pollen on stigma, insect visitation, and visit duration per capitulum were observed. A significantly lower pollen-ovule ratio was observed in the first of six capitula, reflecting higher female functionality. The highest pollination success was found in the second-fourth capitula, whereas florivory increased along the terminal capitula position. High plant density affected the pollen deposited on stigmas via insect visitation and low pollinator visit duration. Different capitula in E. heleocharioides could have different effects: different sexual functionality, enhancement of reproductive output both in quality and quantity through active pollen transfer, and escaping from florivores. High plant density could facilitate outcross-pollen transfer in E. heleocharioides. Multiple perspectives are important for determining potential reproductive success in ex-situ conservation. Thus, density management reflecting capitulum characteristics could improve the efficiency of conservation efforts for E. heleocharioides.

Approximate Bayesian computation analysis of EST-associated microsatellites indicates that the broadleaved evergreen tree Castanopsis sieboldii survived the Last Glacial Maximum in multiple refugia in Japan.

Climatic changes have played major roles in plants' evolutionary history. Glacial oscillations have been particularly important, but some of their effects on plants' populations are poorly understood, including the numbers and locations of refugia in Asian warm temperate zones. In the present study, we investigated the demographic history of the broadleaved evergreen tree species Castanopsis sieboldii (Fagaceae) during the last glacial period in Japan. We used approximate Bayesian computation (ABC) for model comparison and parameter estimation for the demographic modeling using 27 EST-associated microsatellites. We also performed the species distribution modeling (SDM). The results strongly support a demographic scenario that the Ryukyu Islands and the western parts in the main islands (Kyushu and western Shikoku) were derived from separate refugia and the eastern parts in the main islands and the Japan Sea groups were diverged from the western parts prior to the coldest stage of the Last Glacial Maximum (LGM). Our data indicate that multiple refugia survived at least one in the Ryukyu Islands, and the other three regions of the western and eastern parts and around the Japan Sea of the main islands of Japan during the LGM. The SDM analysis also suggests the potential habitats under LGM climate conditions were mainly located along the Pacific Ocean side of the coastal region. Our ABC-based study helps efforts resolve the demographic history of a dominant species in warm temperate broadleaved forests during and after the last glacial period, which provides a basic model for future phylogeographical studies using this approach.

Receptor-type protein tyrosine phosphatase κ directly dephosphorylates CD133 and regulates downstream AKT activation.

Although CD133 has been considered to be a molecular marker for cancer stem cells, its functional roles in tumorigenesis remain unclear. We here examined the molecular basis behind CD133-mediated signaling. Knockdown of CD133 resulted in the retardation of xenograft tumor growth of colon cancer-derived HT-29 and LoVo cells accompanied by hypophosphorylation of AKT, which diminished ß-catenin/T-cell factor-mediated CD44 expression. As tyrosine residues of CD133 at positions 828 and 852 were phosphorylated in HT-29 and SW480 cells, we further addressed the significance of this phosphorylation in the tumorigenesis of SW480 cells expressing mutant CD133, with substitution of these tyrosine residues by glutamate (CD133-EE) or phenylalanine (CD133-FF). Forced expression of CD133-EE promoted much more aggressive xenograft tumor growth relative to wild-type CD133-expressing cells accompanied by hyperphosphorylation of AKT; however, CD133-FF expression had negligible effects on AKT phosphorylation and xenograft tumor formation. Intriguingly, the tyrosine phosphorylation status of CD133 was closely linked to the growth of SW480-derived spheroids. Using yeast two-hybrid screening, we finally identified receptor-type protein tyrosine phosphatase κ (PTPRK) as a binding partner of CD133. In vitro studies demonstrated that PTPRK associates with the carboxyl-terminal region of CD133 through its intracellular phosphatase domains and also catalyzes dephosphorylation of CD133 at tyrosine-828/tyrosine-852. Silencing of PTPRK elevated the tyrosine phosphorylation of CD133, whereas forced expression of PTPRK reduced its phosphorylation level markedly and abrogated CD133-mediated AKT phosphorylation. Endogenous CD133 expression was also closely associated with higher AKT phosphorylation in primary colon cancer cells, and ectopic expression of CD133 enhanced AKT phosphorylation. Furthermore, lower PTPRK expression significantly correlated with the poor prognosis of colon cancer patients with high expression of CD133. Thus, our present findings strongly indicate that the tyrosine phosphorylation of CD133, which is dephosphorylated by PTPRK, regulates AKT signaling and has a critical role in colon cancer progression.

Quantification of atomic-scale elasticity on Ge(001)-c(4 × 2) surfaces via noncontact atomic force microscopy with a tungsten-coated tip.

We investigated the bonding stiffness of individual atoms on substrate surfaces using noncontact atomic force microscopy with frequency modulation. We measured the frequency shift distribution of the (110) plane above buckling-up and buckling-down dimer atoms of the Ge(001)-c(4 × 2) surface using a tungsten-coated atomic force microscopy cantilever. The tip-surface chemical force distribution was reproduced from the frequency shift data using calculations based on Sader's formula. The total harmonic bonding stiffness between the dimer atoms and the substrate was first discovered by fitting the Morse force to the tip-surface chemical force distribution with consideration of the relaxation in the tip-surface gap. By excluding the contribution exerted by the probe tip, we observed that the harmonic bonding compliance of the buckling-up dimer atom was 4.8 × 10(-3) m/N stiffer than that of the buckling-down dimer atom. This technique for probing the elastic bonding states of individual surface atoms at the atomic scale is unique.

CD133 suppresses neuroblastoma cell differentiation via signal pathway modification.

CD133 (prominin-1) is a transmembrane glycoprotein expressed on the surface of normal and cancer stem cells (tumor-initiating cells), progenitor cells, rod photoreceptor cells and a variety of epithelial cells. Although CD133 is widely used as a marker of various somatic and putative cancer stem cells, its contribution to the fundamental properties of cancer cells, such as tumorigenesis and differentiation, remains to be elucidated. In the present report, we found that CD133 was expressed in several neuroblastoma (NB) cell lines/tumor samples. Intriguingly, CD133 repressed NB cell differentiation, for example neurite extension and the expression of differentiation marker proteins, and was decreased by several differentiation stimuli, but accelerated cell proliferation, anchorage-independent colony formation and in vivo tumor formation of NB cells. NB cell line and primary tumor-sphere experiments indicated that the molecular mechanism of CD133-related differentiation suppression in NB was in part dependent on neurotrophic receptor RET tyrosine kinase regulation. RET transcription was suppressed by CD133 in NB cells and glial cell line-derived neurotrophic factor treatment failed to induce RET in CD133-expressing cells; RET overexpression rescued CD133-related inhibition of neurite elongation. Of note, CD133-related NB cell differentiation and RET repression were mainly dependent on p38MAPK and PI3K/Akt pathways. Furthermore, CD133 has a function in growth and RET expression in NB cell line- and primary tumor cell-derived tumor spheres. To the best of our knowledge, this is the first report of the function of CD133 in cancer cells and our findings may be applied to improve differentiation induction therapy for NB patients.

Bmi1 is a MYCN target gene that regulates tumorigenesis through repression of KIF1Bbeta and TSLC1 in neuroblastoma.

Recent advances in neuroblastoma (NB) research addressed that epigenetic alterations such as hypermethylation of promoter sequences, with consequent silencing of tumor-suppressor genes, can have significant roles in the tumorigenesis of NB. However, the exact role of epigenetic alterations, except for DNA hypermethylation, remains to be elucidated in NB research. In this paper, we clarified the direct binding of MYCN to Bmi1 promoter and upregulation of Bmi1 transcription by MYCN. Mutation introduction into an MYCN binding site in the Bmi1 promoter suggests that MYCN has more important roles in the transcription of Bmi1 than E2F-related Bmi1 regulation. A correlation between MYCN and polycomb protein Bmi1 expression was observed in primary NB tumors. Expression of Bmi1 resulted in the acceleration of proliferation and colony formation in NB cells. Bmi1-related inhibition of NB cell differentiation was confirmed by neurite extension assay and analysis of differentiation marker molecules. Intriguingly, the above-mentioned Bmi1-related regulation of the NB cell phenotype seems not to be mediated only by p14ARF/p16INK4a in NB cells. Expression profiling analysis using a tumor-specific cDNA microarray addressed the Bmi1-dependent repression of KIF1Bbeta and TSLC1, which have important roles in predicting the prognosis of NB. Chromatin immunoprecipitation assay showed that KIF1Bbeta and TSLC1 are direct targets of Bmi1 in NB cells. These findings suggest that MYCN induces Bmi1 expression, resulting in the repression of tumor suppressors through Polycomb group gene-mediated epigenetic chromosome modification. NB cell proliferation and differentiation seem to be partially dependent on the MYCN/Bmi1/tumor-suppressor pathways.

Plk1 regulates liver tumor cell death by phosphorylation of TAp63.

We previously found that Plk1 inhibited the p53/p73 activity through its direct phosphorylation. In this study, we investigated the functional role of Plk1 in modulating the p53 family member TAp63, resulting in the control of apoptotic cell death in liver tumor cells. Immunoprecipitation and in vitro pull-down assay showed that p63 binds to the kinase domain of Plk1 through its DNA-binding region. in vitro kinase assay indicated that p63 is phosphorylated by Plk1 at Ser-52 of the transactivating (TA) domain. Plk1 decreased the protein stability of TAp63 by its phosphorylation and suppressed TAp63-induced cell death. Furthermore, Plk1 knockdown in p53-mutated liver tumor cells transactivated p53 family downstream effectors, PUMA, p21(Cip1/WAF1) and 14-3-3sigma, and induced apoptotic cell death. Double knockdown of Plk1/p63 attenuated Plk1 knockdown-induced apoptotic cell death and transactivation. Intriguingly, both Plk1 and p63 are highly expressed in the side population (SP) fraction of liver tumor cells compared to non-SP fraction cells, suggesting the significance of Plk1/TAp63 in the control of cell death in tumor-initiating SP fraction cells. Thus, Plk1 controls TAp63 by its phosphorylation and regulates apoptotic cell death in liver tumor cells. Plk1/TAp63 may be a suitable candidate as a molecular target of liver tumor treatments.

Large cell neuroendocrine carcinoma of the tongue base: case report of an unusual location with immunohistochemical analysis.

A case of large cell neuroendocrine carcinoma (LCNEC) of the tongue base is described. It was characterized by solid tumor nests with central necrosis and rosette formation resembling basaloid squamous cell carcinoma. Immunohistochemical examination revealed that this tumor had neuroendocrine differentiation. It was diagnosed as LCNEC of the tongue base. Pulmonary LCNEC is a well-established entity, but LCNEC also occurs in other organs. This is the first report of mucosal LCNEC in the oral cavity. Basal cells in the normal squamous epithelium around the tumor indicated positivity for neural cell adhesion molecule and N-cadherin. These cells were considered neuroendocrine-related cells in the lingual squamous epithelium, which are related to the tumorigenesis of mucosal LCNEC in the tongue base.

N-MYC promotes cell proliferation through a direct transactivation of neuronal leucine-rich repeat protein-1 (NLRR1) gene in neuroblastoma.

Neuronal leucine-rich repeat protein-1 (NLRR1) gene encodes a type I transmembrane protein with unknown function. We have previously described that NLRR1 gene is highly expressed in unfavorable neuroblastomas as compared with favorable tumors and its higher expression levels correlate significantly with poor clinical outcome. In this study, we have found that NLRR1 gene is one of direct target genes for N-MYC and its gene product contributes to N-MYC-dependent growth promotion in neuroblastoma. Expression levels of NLRR1 were significantly associated with those of N-MYC in various neuroblastoma cell lines as well as primary neuroblastoma tissues. Indeed, enforced expression of N-MYC resulted in a remarkable induction of the endogenous NLRR1. Consistent with these results, we have identified two functional E-boxes within the promoter region and intron 1 of NLRR1 gene. Intriguingly, c-myc also transactivated NLRR1 gene. Enforced expression of NLRR1 promoted cell proliferation and rendered cells resistant to serum deprivation. In support with these observations, small-interfering RNA-mediated knockdown of the endogenous NLRR1-reduced growth rate and sensitized cells to serum starvation. Collectively, our present findings provide a novel insight into understanding molecular mechanisms behind aggressive neuroblastoma with N-MYC amplification.

Stress via p53 pathway causes apoptosis by mitochondrial Noxa upregulation in doxorubicin-treated neuroblastoma cells.

In this study, we employed a panel of cell lines to determine whether p53-dependent cell death in neuroblastoma (NB) cells is caused by apoptotic cellular function, and we further studied the molecular mechanism of apoptosis induced via the p53-dependent pathway. We obtained evidence that a type of p53-dependent stress, doxorubicin (Doxo) administration, causes accumulation of p53 in the nucleus of NB cells and phosphorylation of several serine residues in both Doxo-sensitive and -resistant cell lines. Upregulation of p53-downstream molecules in cells and upregulation of Noxa in the mitochondrial fraction were observed only in Doxo-sensitive NB cells. Significance of Noxa in the Doxo-induced NB cell death was confirmed by Noxa-knockdown experiments. Mitochondrial dysfunction, including cytochrome-c release and membrane potential disregulation, occurred and resulted in the activation of the intrinsic caspase pathway. However, in the Doxo-resistant cells, the accumulation in the nucleus and phosphorylation of p53 did not induce p53-downstream p21(Cip1/Waf1) expression and the Noxa upregulation, resulting in the retention of the mitochondrial homeostasis. Taken together, these findings indicate that the p53 pathway seems to play a crucial role in NB cell death by Noxa regulation in mitochondria, and inhibition of the induction of p53-downstream effectors may regulate drug resistance of NB cells.

DFF45/ICAD restores cisplatin-induced nuclear fragmentation but not DNA cleavage in DFF45-deficient neuroblastoma cells.

We have previously defined a homozygously deleted region at chromosome 1p36.2-p36.3 in human neuroblastoma cell lines, NB-1 and NB-C201, and identified six genes including DFF45/ICAD within this region. In this study, we found that NB-C201 cells are much more resistant to various genotoxic stresses such as cisplatin (CDDP) than CHP134 and SH-SY5Y cells that do not have the homozygous deletion. To examine a role(s) of DFF45 in the regulation of apoptosis in response to CDDP, we have established stably DFF45-expressing NB-C201 cell clones (DFF45-1 and DFF45-3) and a control cell clone (NB-C201-C) using a retrovirus-mediated gene transfer. In contrast to NB-C201-C cells, DFF45-3 cells displayed apoptotic nuclear fragmentation in response to CDDP. Although CDDP-induced proteolytic cleavage of procaspase-3 and DFF45 in DFF45-3 cells, we could not detect a typical apoptotic DNA fragmentation. Additionally, deletion analysis revealed that C-terminal region of DFF45 is required for inducing nuclear fragmentation. Unexpectedly, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays demonstrated that DFF45 has undetectable effect on CDDP sensitivity of NB-C201 cells. Taken together, our present results suggest that DFF45/DFF40 system may be sufficient for CDDP-induced nuclear fragmentation but not DNA cleavage.

Chromosomal change during 6-mercaptopurine (6-MP) therapy in juvenile myelomonocytic leukemia: the growth of a 6-MP-refractory clone that already exists at onset.

Among 11 JMML children, two had an abnormal karyotype, and nine had a normal karyotype at onset. In one patient with trisomy 8 and four patients with a normal karyotype, a new clone with an aberrant karyotype emerged 1-14 months after 6-mercaptopurine (6-MP) therapy as shown by G-banding analyses. Fluorescence in situ hybridization disclosed that an abnormal clone existed in approximately 3-6% of bone marrow cells at onset or before 6-MP therapy in all the four cases examined, and increased to approximately 12-90% during the treatment. In culture with granulocyte-macrophage colony-stimulating factor, cytogenetically abnormal clones that proliferated during 6-MP therapy possessed significantly less sensitivity to the antimetabolite, compared with cells that decreased in numbers after the therapy. A PTPN11 mutation was detected in all of granulocyte-macrophage colonies irrespective of karyotypic aberration in one patient, whereas approximately 80% of erythroid colonies and 20% of mixed colonies possessed neither a PTPN11 mutation nor chromosomal abnormalities. The appearance of chromosomal aberrations shown by G-banding during 6-MP therapy in some JMML cases may result, in part, from the growth of a 6-MP-refractory clone that already exists at onset. It is possible that treatment with 6-MP promotes progression of the disease.

Engineered IGF-I expression induces glandular enlargement in the murine prostate.

IGF-I has been implicated as a factor that may predispose one to prostate cancer and to benign prostatic hypertrophy (BPH). We established murine IGF-I transgenic mice under the control of rat probasin promoter and analysed the histology of the murine IGF-I-overexpressing prostate. Immunohistochemically, IGF-I was expressed in prostatic epithelial cells or basement membranes of the ventral, dorsal and lateral lobes in a line of IGF-I transgenic mice, but not in their control littermates. The anterior lobe did not express IGF-I. IGF-binding protein-3 (IGFBP-3), inhibitory to the mitogenic action of IGF-I, was detected in epithelial cells of prostatic ventral lobes, but not in those of the dorsal, lateral or anterior lobes of IGF-I transgenic mice. In controls, IGFBP-3 was not detected in epithelial cells of any prostatic lobe. Macroscopic prostatic size and the appearance of IGF-I transgenic mice were comparable with those of their control littermates of the same age. With a computed morphometric analysis, epithelial glands and intraglandular lumens in the prostatic lobes except the ventral lobe were smaller at 17 Months of age than at 14 Months both in IGF-I transgenic mice and controls. Glands and intraglandular lumens in the ventral prostatic lobes of IGF-I transgenic mice expressing more IGF-I protein in the prostate than controls were dense and enlarged similar to cysts compared with those of non-transgenic littermates without showing epithelial growth. Glands and lumens in the dorsal and lateral lobes of the IGF-I transgenic mice were also larger than controls at 14 and/or 17 Months of age. Glands in the anterior prostatic lobe of the IGF-I transgenic mice were not morphologically or morphometrically different from those of non-transgenic littermates. In conclusion, IGF-I transgenic mice under the control of rat probasin promoter showed more dense and enlarged epithelial glands in their prostatic ventral, dorsal and lateral lobes.

Fatal hemophagocytic syndrome after living-related liver transplantation: a report of two cases.

BACKGROUND: Hemophagocytic syndrome (HPS) is a serious hematological disorder caused by activated T lymphocytes in immunologically compromised patients. There is no report of HPS in liver transplant recipients. METHODS: Among 135 patients who underwent living-related liver transplantation between June 1990 and October 2000, HPS developed in two pediatric patients (1.5%) on the 15th and 134th postoperative day, respectively. The courses of these patients were evaluated. RESULTS: The cause of HPS was unknown in patient 1 and suspected to be Epstein-Barr virus infection in patient 2. The course of patient 2 was also complicated by posttransplant lymphoproliferative disorder. Both patients had high fever, pancytopenia, coagulopathy, and marked elevation of serum-soluble interleukin 2 receptor, serum ferritin, and urine beta2-microglobulin levels. The diagnosis was established based on clinical findings, laboratory data, and bone marrow biopsy. Both patients died in an acute course despite intensive care. CONCLUSIONS: HPS should be recognized as a severe hematological complication in liver transplant patients. Prompt institution of adequate treatment is necessary to prevent fatality.

Prostate-specific antigen induces osteoplastic changes by an autonomous mechanism.

The high prevalence of osteoplastic bone metastasis in prostate cancer (PC) is believed to be attributable to the production of osteoblast-stimulating factors by PC cells. Prostate-specific antigen (PSA) is a serine protease and an important serological marker for PC. Exposure of osteoblasts to PSA in vitro was found to result in cell proliferation and marked upregulation of transforming growth factor-beta (TGF-beta) mRNA expression. This PSA-induced increase in osteoblast proliferation was inhibited by anti-TGF-beta antibodies and serine protease inhibitors. In vivo, PSA markedly enhanced osteoplastic changes in human adult bone implanted into NOD/SCID mice without PC cells, and alpha(1)-antichymotrypsin prevented the PSA-induced increase in bone volume. PSA promotes osteoplastic change by activating an osteoblast autonomous mechanism that is independent of the production of bone growth factors by PC cells.

Microvessel density predicts the radiosensitivity of metastatic head and neck squamous cell carcinoma in cervical lymph nodes.

Cervical lymph node metastasis is the most common recurrence pattern of head and neck squamous cell carcinoma (HNSCC), and it is usually treated with radiation therapy and/or neck dissection. There has long been a desire for markers useful in predicting radiosensitivity to enable assignment of patients with recurrent head and neck cancer to clinical trials to improve their survival rates and quality of life. A total of 43 cases of HNSCC treated with whole or elective neck irradiation (total dose, 26-70 Gy; median, 60 Gy) for recurrent metastatic SCC in neck lymph nodes after neck dissection between 1992 and 1999 were the subject of this study. The relationship between radiosensitivity and clinicopathological and histopathological factors, including the Ki-67-labeling index for cell proliferation, p53 immunoreactivity and microvessel density (MVD), in surgical neck lymph node specimens were investigated by univariate and multivariate analysis. Of the 43 patients, 31 had recurrent tumors in neck lymph nodes after radiotherapy. Univariate analysis revealed significant associations between radiosensitivity and both high grade of keratinization (p=0.033) and low MVD (p=0.004), and marginally significant associations between radiosensitivity and grade of differentiation of the cancer in the lymph nodes (p=0.070). Multivariate analysis showed that only MVD had predictive value (p=0.016). Tumors with a high MVD possessed a significantly better neck control rate than tumors with a low MVD (p=0.004) by Kaplan-Meier analysis. MVD can be used as a good predictive marker for radiosensitivity of metastatic HNSCCs in cervical lymph nodes after neck dissection.

Effect of cernitin pollen-extract on experimental nonbacterial prostatitis in rats.

BACKGROUND: The treatment for chronic nonbacterial prostatitis (NBP) has not been established. Cernitin pollen-extract (CN-009) is reported to have therapeutic effects for NBP. The effects and mechanisms of CN-009 were investigated. METHODS: Ten-month-old rats were used with administration of estradiol after castration, which were similar to human NBP histologically. Since CN-009 consists of T-60 and GBX, these drugs were administered, respectively. The prostate was evaluated histopathologically including glandular damage (epithelial score), stromal ratio and immunohistochemical assays for epithelial function (PAP), stromal evaluation (Vimentin), cell proliferation (PCNA) and apoptosis (deoxyuridine triphosphate biotin nick end-labeling (TUNEL)). RESULTS: Controls revealed severe acinar gland atrophy and stromal proliferation. CN-009 showed diminished these damages. Epithelial score was better (P < 0.01) and PAP positive materials were more abundant in CN-009 and GBX than in Controls. The stromal ratio was lower in CN-009 (P < 0.01) and T-60 (P < 0.05). There was no difference for PCNA positive cells in the epithelium and stroma, and TUNEL positive cells in epithelium. While, the number of TUNEL positive cells in the stroma of CN-009 and T-60 increased (P < 0.01). CONCLUSIONS: These findings suggest that CN-009 protects acinar epithelial cells mainly by GBX and also inhibits stromal proliferation in association with enhanced apoptosis mainly by T-60.

Dynamic DNA methylation change in the CpG island region of p15 during human myeloid development.

We examined the kenetics of p15 methylation and expression during myeloid development. We treated human cord blood CD34+ cells with either GM-CSF alone or in combination with stem cell factor and followed methylation at this locus using bisulfite genomic sequencing. CD34+ cells were found to be either fully methylated or completely unmethylated at 27 CpG dinucleotide sites in exon 1 and at 18 CpG sites in the promoter region of the p15 gene. A time-course study showed that the percentage of the allelic methylation of p15 CpG island increased to approximately 50% to 60% until 7 days after cytokine stimulation, then decreased to less than 10% after 21 days. The methylation was also observed in bone marrow CD34+ cells exposed to GM-CSF. p15 expression varied inversely with methylation. Expression was negligible or at low levels until 14 days, after which it increased substantially. The frequency of myeloid colony-forming cells in the progeny decreased and myeloid-specific markers increased in the later stages. Based on our observations on cells grown with GM-CSF and 5-aza-2'-deoxycytidine, DNA methylation of the p15 promoter region CpG island appears to be associated with proliferation rather than differentiation of normal human myeloid progenitors.

Image analysis of microvessel surface area predicts radiosensitivity in early-stage laryngeal carcinoma treated with radiotherapy.

PURPOSE: The tissue oxygenation level, which is theoretically governed by distance from blood vessels, is one of the most important modulators of the radiosensitivity of carcinoma. A computed image analysis system for the detection of tissue oxygenation was developed to establish a method of predicting radiosensitivity in early-stage laryngeal carcinoma treated by curative radiotherapy. EXPERIMENTAL DESIGN: Microvessel structures labeled with CD31 antigen were investigated in 55 patients undergoing curative radiotherapy for T1 and T2 laryngeal carcinoma. We calculated (a) microvessel density [(MVD) vessels/field] under a microscope; (b) the ratio of the total microvessel number (TN):tumor area (TA) [TN:TA; vessels/mm2]; (c) the ratio of the total microvessel perimeter (TP):TA (TP:TA; mm/mm2); and (d) the ratio of tumor tissue area >150 microm from microvessels (hypoxic ratio; %) as parameters of tissue oxygenation in each whole biopsy specimen by using an image analyzer. We compared each of these factors with radiosensitivity. RESULTS: Mann-Whitney's U test revealed that tumors with a high MVD (median, 42 vessels/field), high TN:TA ratio (median=40.9 vessels/mm2), high TP:TA ratio (median, 2.92 mm/mm2), and low hypoxic ratio (median, 30.3%) had significantly greater radiosensitivity than tumors with a low MVD, low TN:TA ratio, low TP:TA ratio or high hypoxic ratio (P = 0.002, P = 0.0004, P < 0.0001, and P = 0.004, respectively). CONCLUSIONS: Prediction of radiosensitivity on the basis of the TP:TA ratio can be used as an efficient means of avoiding ineffective radiation, complications after salvage surgery, and prolonged hospital stays.

Induction of apoptosis by cyclooxygenase-2 inhibitors in prostate cancer cell lines.

Prostaglandins are thought to play an important role in the proliferation of prostate cancer and are highly expressed in prostate cancer tissue. Cyclooxygenase-2 (COX-2), or prostaglandin endoperoxide synthase, is a key enzyme in the conversion of arachidonic acid into prostaglandin. In several cancers, COX-2 contributes to the proliferation and metastasis of cancer cells. To assess the role of COX-2 in prostate cancer, we investigated whether the inhibition of COX-2 affected the proliferation of prostate cancer cells. The human prostate cancer cell lines, LNCaP and PC 3, and a normal prostate stromal cell line (PrSC) were treated with COX-2 inhibitors NS 398 and Etodolac. The proliferation rate of the cell lines was examined using 3(4,5-dimethylethiazoly 1-2-) 2,5-diphonyl tetrazolium bromide (MTT) assays. A DNA fragmentation assay was also used for proof of apoptosis. COX-2 inhibitors could suppress the proliferation of LNCaP and PC 3 cells. In contrast, PrSC was not affected by COX-2 inhibitors. These suppressive effects occurred in a time- and dose-dependent manner. One of mechanisms responsible for cell death was apoptosis. COX-2 seems to play a significant role in the progression of prostate cancer. COX-2 may be a therapeutic target for prostate cancer. Since COX-2 inhibitors suppress proliferation and induce apoptosis in prostate cancer cells, and have no effect in normal prostate stromal cells, COX-2 inhibitors will be useful for the treatment of prostate cancer.