[Comparison of dose distribution on radiographic film and radiochromic film for intensity modulated radiation therapy].

PURPOSE: Radiographic film is generally used for inspection of dose distribution in intensity modulated radiation therapy (IMRT) at many institutions. However, the distribution of filmless systems can be expected to be used increasingly in the future. Therefore, we confirmed the utility of radiochromic film by comparing it with radiographic film that does not need an automatic processor. RESULT: Difference in does measured by radiographic film and radiochromic film tended to increase in the low does area, but it was limited in a range of 1.5%. CONCLUSION: When the dose distribution was verified in a highly accurate radiation therapy such as IMRT, the results suggested that radiochromic film can be useful in addition to radiographic film.

Different HATS of the ING1 gene family.

The ING family of proteins are involved in chromatin remodelling, and bind to and affect the activity of histone acetyltransferase, histone deacetylase, and factor acetyltransferase protein complexes. Some family members affect transcription, including the expression of p53-inducible genes such as p21 and Bax, and ING2 induces p53 acetylation on a site implicated in the regulation of p53 activity. ING1 promotes DNA repair and interacts with proliferating cell nuclear antigen, thus linking DNA repair, apoptosis and chromatin remodelling. Here, we summarize what is known about the molecular interactions of ING1 family proteins and, based on these interactions, develop a model to better understand the impact of ING proteins on multiple biological processes.

Expression of estrogen receptor beta wild-type and its variant ERbetacx/beta2 is correlated with better prognosis in breast cancer.

BACKGROUND: The clinical value of estrogen receptor (ER) beta and its variants has been investigated. However, reported results have frequently been discordant. METHODS: We investigated mRNA and protein expression of ERbeta wild type (ERbeta1) and its variant, ERbetacx/beta2, by quantitative real-time reverse transcriptase-polymerase chain reaction and immunohistochemistry in 150 breast cancer tissues, and analyzed association between their expression and clinicopathological factors and prognosis. RESULTS: ERbeta1 mRNA expression was significantly correlated with progesterone receptor expression and low histological grade. ERbeta1 protein expression was significantly correlated with small tumor size, negative lymph node status and low histological grade. ERbetacx/beta2 protein expression was significantly correlated with ERalpha expression and low histological grade. Patients with high expression of ERbeta1 or ERbetacx/beta2 had a significantly better disease-free and overall survival than those with low expression. In multivariate analysis, ERbetacx/beta2 mRNA expression was identified as a prognostic factor in disease-free and overall survival. CONCLUSION: Our results indicate that ERbetacx/beta2 mRNA expression is an independent prognostic factor in breast cancer. ERbeta expression status, both wild-type and the variant cx/beta2, might represent significant predictors of breast cancer prognosis.

NCOR1 mRNA is an independent prognostic factor for breast cancer.

The transcriptional function of estrogen receptor alpha (ERalpha) can be modulated by co-regulatory proteins. In the present study, therefore, the level of expression of one of the co-regulator Nuclear Receptor Co-repressor 1 (NCOR1) mRNA has been assessed by quantitative real-time RT-PCR in 160 cases of invasive breast carcinoma. It was found that NCOR1 mRNA was expressed at significantly higher levels in patients over 50 years of age, without axillary lymph node involvement, with tumor size less than 2 cm, with low or intermediate histological grade, with ERalpha/PgR-positive and with HER2 negative tumors. Patients with high levels of expression of NCOR1 mRNA have a better prognosis than those with low expression. Univariate and multivariate prognostic analysis demonstrated that NCOR1 mRNA is an independent prognostic factor for breast cancer.

Clinical usefulness of oral combination chemotherapy of 5'-deoxy-5-fluorouridine (5'-DFUR) and cyclophosphamide for metastatic breast cancer.

BACKGROUND: It has been reported that 5'-deoxy-5-fluorouridine (5'-DFUR), the pro-drug of 5-FU, is effective treatment for breast cancer that express thymidine phosphorylase (dThdPase). Since oral cyclophosphamide (CPA) induces dThdPase, a synergistic effect can be expected by combining CPA with 5'-DFUR. We evaluated the usefulness of combination chemotherapy using CPA and 5'-DFUR in patients with relapsed breast cancer in this prospective phase II study. METHODS: Patients with relapsed, advanced breast cancer with evaluable lesions were given 5'-DFUR at 800 mg/day/body and CPA at 100 mg/day/body for 2 weeks, then underwent 2 weeks of drug withdrawal. This was considered one course of treatment. It was repeated until progressive disease (PD) was confirmed. The lesions were evaluated according to UICC criteria and compared with regard to the clinical status. RESULTS: Sixty-four patients with relapsed, advanced breast cancer were registered. Complete response (CR) was seen in 7 patients, partial response (PR) in 12 patients, no change (NC) in 25 patients, of whom 11 achieved long NC with the effect lasting for more than 6 months, and PD was seen in 20 patients. The response rate was 29.7%. The total number of CR, PR, and long NC cases was 30, which comprise-46.9% of the total 64 cases (the clinical benefit rate). As for adverse events, hematological toxicities were seen in 9 patients, with grade 3 toxicits was seen in 1 patient. All other adverse events were grade 1 or 2. CONCLUSION: For those patients who achieved an effect more than NC, it was possible to continue the therapy for an average of 53 weeks. This treatment method is worth considering for patients who have metastatic breast cancer, that is not life threatening.

ING1 isoforms differentially affect apoptosis in a cell age-dependent manner.

Recently, several novel human ING1 isoforms have been cloned. However,the biochemical functions and the involvement of these proteins in apoptosis remain uncharacterized. We have examined the apoptotic effects and biochemical functions of the two major human ING1 isoforms p47(ING1a) and p33(ING1b) in young and senescent human diploid fibroblasts induced to enter into apoptosis by diverse treatments. We have found that ING1 displayed isoform-, stimulus- and cell age-dependent apoptotic properties. We present evidence indicating that ING1 proteins bind to chromatin and are regulated in a manner related to their apoptotic properties. In agreement with previous reports, we have found that only young but not senescent fibroblasts were able to enter into apoptosis induced by growth factor deprivation. This effect was accompanied by up-regulation of endogenous p33(ING1b). Ectopic up-regulation of p33(ING1b), but not p47(ING1a), also induced apoptosis and sensitized young but not senescent cells to UV irradiation and hydrogen peroxide-mediated apoptosis. Cotransfection of p33(ING1b) and the tumor suppressor p53 increased the percentage of apoptotic cells yielded by either of these two proteins alone, in agreement with data from tumor cell models. Finally, we found that the chromatin binding affinity of p33(ING1b) was increased in senescent cells, which were resistant to apoptosis. Together, these data support the idea that the apoptotic functions of ING1 may be exerted by chromatin-related functions that are subject to cell age-dependent mechanisms of regulation.

ING1 represses transcription by direct DNA binding and through effects on p53.

The ING family of proteins is involved in the regulation of diverse processes ranging from cell cycle and cellular senescence to apoptosis. These effects are most likely through activation of acetylation-dependent pathways that ultimately alter gene expression. Despite reports linking ING to p53 activation, the molecular basis of how ING activates p53 function has not been elucidated. In this study, we found that a subset of ING family members strongly repressed human alpha-fetoprotein (AFP) promoter activity but stimulated the p21(WAF1) promoter in parallel experiments in the same cell type, similar to the effects of p53. The p47(ING1a) isoform also repressed AFP promoter activity, but in contrast to other ING isoforms, it repressed the p21(WAF1) promoter. p47(ING3) up-regulated p21(WAF1) promoter activity, but it did not have any effect on the AFP promoter. ING1b and ING2 also repressed the AFP promoter in Hep3B p53-null cell lines, and p53 coexpression enhanced this transcriptional repression. Suppression of AFP gene transcription by ING was strongly dependent on AT-motifs that bind to the hepatocyte nuclear factor 1 (HNF1) transcription factor. Indeed, electrophoretic mobility shift assays confirmed that HNF1 binds to AT-motifs, but we found, surprisingly, that the ING1 complexes binding to these AT-motifs were devoid of HNF1 protein. Both ING1 and p53 were able to suppress AFP transcription and cause p21 induction; hSIR2, a negative regulator of the p53 protein, showed the opposite effects on the AFP promoter and, like HDAC1, repressed p21 promoter activity. In addition, we found that p33(ING1b) physically interacts with hSIR2, reverses its ability to induce the AFP promoter, and induces acetylation of p53 residues at Lys(373) and/or Lys(382). These findings provide novel evidence that p33(ING1b) represses AFP transcription by at least two mechanisms, one of which includes p53. The first is by binding to the AT-motif and excluding HNF1 binding while possibly targeting HAT activity to promoter regions, and the second is by increasing the levels of active, acetylated p53 via binding and inhibiting the ability of hSIR2 to deacetylate p53 protein.

Oxidation and interaction of DJ-1 with 20S proteasome in the erythrocytes of early stage Parkinson's disease patients.

Parkinson's disease (PD) is a progressive, age-related, neurodegenerative disorder, and oxidative stress is an important mediator in its pathogenesis. DJ-1, the product of the causative gene of a familial form of PD, plays a significant role in anti-oxidative defence to protect cells from oxidative stress. DJ-1 undergoes preferential oxidation at the cysteine residue at position 106 (Cys-106) under oxidative stress. Here, using specific antibodies against Cys-106-oxidized DJ-1 (oxDJ-1), it was found that the levels of oxDJ-1 in the erythrocytes of unmedicated PD patients (n = 88) were higher than in those of medicated PD patients (n = 62) and healthy control subjects (n = 33). Elevated oxDJ-1 levels were also observed in a non-human primate PD model. Biochemical analysis of oxDJ-1 in erythrocyte lysates showed that oxDJ-1 formed dimer and polymer forms, and that the latter interacts with 20S proteasome. These results clearly indicate a biochemical alteration in the blood of PD patients, which could be utilized as an early diagnosis marker for PD.

Quantitative determination, by real-time reverse transcription polymerase chain reaction, of aromatase mRNA in invasive ductal carcinoma of the breast.

BACKGROUND: Estrogen is a mitogenic factor that is implicated in the genesis and progression of breast cancer via its binding to estrogen receptor (ER)-alpha. Synthesis of estrogen in situ is believed to be catalyzed mainly by aromatase. Previous studies comparing the relative contributions from tumor cells and stromal cells to local estrogen synthesis, as assessed by immunohistochemical analysis, were quite controversial and no consistent relationship was found between the presence of aromatase and any clinicopathologic factor. In addition, previous studies into aromatase gene expression and clinicopathologic factors are limited. METHODS: We assessed the level of expression of aromatase mRNA, using quantitative real-time RT-PCR, in 162 cases of invasive ductal carcinoma of the breast. Associations between aromatase expression and different clinicopathologic factors were sought. RESULTS: It was found that aromatase mRNA was expressed at significantly higher levels in patients older than 50 years, in those without axillary lymph node involvement, in those with tumor size less than 2 cm, and in ER-alpha positive tumors. However, no relationship was found between aromatase mRNA expression and any other clinicopathologic factor, including histologic grade and progesterone receptor status. Patients with high levels of expression of aromatase mRNA tended to have a better prognosis than did those patients with low expression. CONCLUSION: These findings imply that ER-alpha and aromatase may be coexpressed in endocrine responsive patients. They may also indicate that aromatase expression could be a marker of endocrine responsiveness, and it may have prognostic implications for breast cancer progression.

Reduced expression of the Syk gene is correlated with poor prognosis in human breast cancer.

Syk is a non-receptor type of protein-tyrosine kinase that is widely expressed in hematopoietic cells. Syk expression has been reported in breast tissue. However, the function of Syk in breast tissue remains unclear. In 25 (28%) of the 90 paired samples of human breast cancer and the matched, adjacent non-cancerous tissues examined here, reduced mRNA expression of Syk in the breast cancer tissues was observed by real-time quantitative reverse transcription-polymerase chain reaction. Eight (32%) of the 25 cases showing reduced Syk mRNA expression demonstrated distant metastasis of breast cancer, while only 4 (6%) of the 65 cases who did not show reduced Syk expression had distant metastasis. Kaplan-Meier curves demonstrated that patients with reduced Syk expression were at significantly increased risk for distant metastasis (P=0.0003). Our data suggest that reduced Syk expression in breast cancers is associated with distant metastasis and poor prognosis; therefore, Syk is considered to be a potential tumor suppressor and anti-metastasis gene in human breast cancer.

ING1 and p53 tumor suppressor gene alterations in adenocarcinomas of the esophagogastric junction.

The aim of this study was to characterize molecular alterations of the recently reported candidate tumor suppressor gene, ING1, and to explore the relationship between ING1 and p53 in a well-defined series of adenocarcinomas of the esophagogastric junction (AdEGJ). Polymerase chain reaction (PCR)-based assays were used to characterize ING1 and p53 alterations, relative to histologically normal esophageal mucosa. Two tumors were found to have ING1 mutations: one novel missense mutation (AGC(Ser)-->ATC(Ile)) at codon 147, and one silent mutation (TCG(Ser)-->TCA(Ser)) at codon 173. Reduced expression of the two major alternatively spliced ING1 messenger RNA variants, p47(ING1a) and p33(ING1b) was variable, but was reduced (1.2-10-fold) in 12 of 19 AdEGJs compared to normal esophageal epithelium. No association between p53 and ING1 alterations was apparent. We conclude that reduced ING1 expression is frequently associated with AdEGJ tumorigenesis, further supporting its role as a tumor suppressor gene, and that ING1 expression is independent of p53 status.

p33(ING1b) stimulates the transcriptional activity of the estrogen receptor alpha via its activation function (AF) 2 domain.

The ING1 gene was originally cloned as a candidate tumor suppressor of human breast cancer, and recent studies suggest that ING1 proteins are involved in chromatin remodeling functions via physical association with both histone acetyltransferases (HATs) and histone deacetylases (HDACs). In this study, we investigated whether p33(ING1b), one of the major ING1 isoforms, modulated the transcriptional activity of estrogen receptor (ER) alpha. In Cos-7 cells transfected with increasing concentrations of a mammalian expression vector encoding for p33(ING1b), estrogen-induced ERalpha transcriptional activity was found to increase in a dose-dependent manner. As p33(ING1b) expression levels increased, transcription of an ER-responsive reporter gene by either estrogen-inducible full-length ERalpha or activation function (AF) 1 deletion mutant was enhanced, while the AF2 deletion mutant was unaffected by the presence of p33(ING1b). These results showed that p33(ING1b) enhanced estrogen-induced ERalpha activity through the AF2 domain. Our data also demonstrated that the antiestrogens inhibited the transcriptional activity of ERalpha as stimulated by p33(ING1b). Furthermore, a weak physical association was observed between in vitro translated p33(ING1b) and ERalpha. Our data presented here demonstrate that p33(ING1b) acts like a coactivator for ERalpha and stimulates estrogen-induced ERalpha transcriptional activity consistent with a function for p33(ING1b) in chromatin remodeling.

Estrogen receptor alpha mutation (A-to-G transition at nucleotide 908) is not found in different types of breast lesions from Japanese women.

BACKGROUND: An estrogen receptor (ER) alpha variant with an A-to-G transition at nucleotide 908 (A908G) has been identified in typical hyperplastic mammary epithelium. This somatic mutation, which induces a Lys-to-Arg substitution at residue 303 within exon 4 at the border of the hinge and hormone-binding domains of the ER alpha, shows increased sensitivity to estrogen compared with wild-type ER alpha. METHODS: To investigate whether this mutation was present in breast lesions, we performed mutation analyses using the PCR-restriction fragment length polymorphism (RFLP) method in 7 cases of hyperplasia, 18 cases of benign breast tumor, 26 cases of ductal carcinoma in situ (DCIS) and 215 cases of invasive ductal carcinoma (IDC). When we extracted DNA, the hyperplastic epithelium and cancer cells of DCIS were microdissected. We also performed direct sequencing analysis from 7 randomly-selected representative cases of hyperplasia, 9 cases of benign breast tumors, 11 cases of DCIS and 20 cases of IDC. RESULTS: No A908G mutation was found in the ER alpha gene in these breast lesions. CONCLUSION: This mutation might be absent or occur in so few cells as to be undetectable by PCR-RFLP or direct sequencing.

Radiosensitivity uncertainty evaluation for the in vitro biophysical modeling of EMT6 cells.

BACKGROUND/AIM: The aims of this study were to evaluate the cell survival uncertainty distribution of radiation and to assess the accuracy of predictions of tumor response by using three different in vitro experimental cell cultures with radiosensitizers (including etanidazole). MATERIALS AND METHODS: Using EMT6 cells and X-rays, the cell survival fraction was obtained from 15, 34, and 21 different experiments under normoxic, hypoxic, and hypoxic-plus-radiosensitizer culture, respectively. RESULTS: The α coefficients were 0.257 ± 0.188, 0.078 ± 0.080, and 0.182 ± 0.116 Gy(-1), respectively. The ß coefficients were 0.0159 ± 0.0208, 0.0076 ± 0.0113, and 0.0062 ± 0.0077 Gy(-2), respectively. The α coefficient and the dose that killed half of the clonogens population (D50) were significantly different between normoxic cell and hypoxic cell cultures (p<0.01), respectively. The use of radiosensitizers under hypoxic conditions improved radiosensitivity. CONCLUSION: Our results suggest that parameter value distributions are required for biophysical modeling of applications for radiotherapy.

[Impact of multileaf collimator leaf positioning accuracy on intensity modulated radiation therapy].

In multileaf collimator (MLC)-based intensity modulated radiation therapy (IMRT), the dose is influenced by the uncertainty of MLC driving control. In this study, we examined the influence of MLC driving control accuracy on dose evaluation (gamma analysis) by evaluating 60-day MLC driving control accuracy (stationary positioning accuracy and positioning reproducibility) once a week as well as measuring IMRT dose distribution. The MLC positioning accuracy accompanied variation over time and tended to expand by 0.1 to 0.15 mm in one week and about 1 mm in 60 days. In terms of reproducibility, errors were within 0.2 mm for more than 95%. For prostate IMRT, when MLC stationary positioning accuracy was around 1 mm, no significant difference was observed in the pass rate in gamma analysis. Therefore, the results suggest that regular maintenance by setting a permissible value determined by the MLC positioning accuracy test can be an effective indicator in the future for maintaining the safety of IMRT.

Use of a diagnostic positron emission tomography-computed tomography system for planning radiotherapy positioning: distortion of the tabletop.

PURPOSE: The aim of this study was to evaluate distortion of the tabletop in a diagnostic positron emission tomography-computed tomography (PET-CT) system to determine its suitability for planning radiotherapy positioning. MATERIALS AND METHODS: Distortion of the tabletop was compared among PET-CT, lineac CT, and CT simulator systems. A phantom or angiography catheter was fixed to the tabletop and imaged after iron plate weight loading. The acquired images were analyzed using radiotherapy planning software. Distortion of the tabletop was measured based on the displayed coordinates. RESULTS: Sinking represented the greatest distortion of the tabletop in all systems. Using the same baseline, the maximum sinking were -0.4, -0.2, and +0.4 cm, respectively. The distortion of the tabletop in the PET-CT system was more similar to that in the lineac CT than in the CT simulator system. CONCLUSION: Distortion of the tabletop in a diagnostic PET-CT system may be within the acceptable range to allow its use for planning radiotherapy positioning.

Preparation and application of monoclonal antibodies against oxidized DJ-1. Significant elevation of oxidized DJ-1 in erythrocytes of early-stage Parkinson disease patients.

DJ-1 was initially identified as a novel oncogene and has recently been found to be a causative gene for a familial form of Parkinson's disease (PD), viz, PARK7. Cysteine residue at position 106 (Cys-106) in DJ-1 was found to be oxidized preferentially under oxidative stress. In the present study, we developed specific antibodies against Cys-106-oxidized DJ-1 using baculovirus particles displaying the surface glycoprotein gp64-fusion protein as the immunizing agent. Western blot analysis combined with two-dimensional gel electrophoresis revealed that these antibodies specifically recognized oxidized DJ-1. Furthermore, we developed a competitive enzyme-linked immunosorbent assay (ELISA) for detecting oxidized DJ-1 and measured blood levels of oxidized DJ-1 in PD patients (n=15). It was observed that the levels of oxidized DJ-1 in erythrocytes of unmedicated PD patients were markedly higher without overlap than those of medicated PD patients and healthy subjects. No significant difference was observed in DJ-1 levels between mediated and unmediated PD patient. These results suggest the oxidative modification of DJ-1 in PD patients and the potential application of the antibody for diagnosis of PD at early-stage.

Clinical analysis of paraneoplastic encephalitis associated with ovarian teratoma.

BACKGROUND: Recently, paraneoplastic encephalitis associated with ovarian teratoma has been described and related to an autoantibody. METHODS: We describe four patients with ovarian teratoma-associated encephalitis (OTE) and compared their clinical pictures with those of 17 previously reported patients with OTE. RESULTS: Clinically, OTE was characterized by the development of acute prominent psychiatric symptoms (20 of 21 patients), seizures (15 of 21 patients), and central hypoventilation (13 of 21 patients). Our patients had hypersalivation (three patients) and cardiac conduction problems (all patients); hypothermia was present in one patient. The mean time from the onset of OTE to tumor diagnosis was 19.6+/-22.1 weeks. Ventilatory support was required for 54.9+/-25.4 days on average. The white blood cell count in cerebrospinal fluid was 55.1+/-61.2/mm3. Twelve patients showed abnormalities on cranial MRI, involving areas such as the temporal regions (seven patients) or brainstem (four patients). In addition to tumor resection, 17 patients received some type of immunotherapy: 17 patients received corticosteroids, 10 received intravenous immunoglobulins, two received cyclophosphamide, seven received plasma exchange. Eighteen patients with OTE had neurological improvement, including 11 with full recovery. CONCLUSIONS: OTE presents with cardiac conduction problems and hypersalivation in addition to psychiatric symptoms, seizures, and central hypoventilation.

Quantitation of HDAC1 mRNA expression in invasive carcinoma of the breast*.

Estrogen is well-established as a mitogenic factor implicated in the tumorigenesis and progression of breast cancer via its binding to the estrogen receptor alpha (ERalpha). Recent data indicate that chromatin inactivation mediated by histone deacetylation (HDAC) and DNA methylation is a critical component of ERalpha silencing in human breast cancer cells. The aim of this study was to determine the expression of the HDAC1 gene in malignant human breast tissue and to correlate our observations with available clinical information. In the present study, the level of expression of HDAC1 mRNA was assessed by LightCycler-based quantitative real-time reverse transcriptase (RT)-PCR analysis in 162 cases of invasive carcinoma of the breast. Associations between HDAC1 mRNA expression and different clinicopathological factors were sought. It was found that HDAC1 mRNA was expressed at significantly higher levels in tumors from patients over 50 years of age and in those tumors without axillary lymph node involvement, that are less than 2 cm, that are of a non-high histological grade, that are HER2 negative and that are ERalpha/PgR positive. Patients with tumors displaying high levels of HDAC1 mRNA expression tended to have a better prognosis in terms of both disease-free and overall survival. However, univariate and multivariate analysis did not show HDAC1 mRNA expression level to be an independent prognostic factor for either disease-free or overall survival. These results imply that HDAC1 mRNA expression could have potential as an endocrine response marker and may have prognostic implications for breast cancer progression.