Serine protease Omi/HtrA2 targets WARTS kinase to control cell proliferation.

The serine protease Omi/HtrA2 was initially regarded as a proapoptotic molecule that proteolyses several proteins to induce cell death. Recent studies, however, indicate that loss of Omi protease activity increases susceptibility to stress-induced cell death. These complicated findings suggest that the protease activity of Omi is involved not only in apoptosis but also in cellular homeostasis. However, the targets which Omi uses to mediate this novel process are unknown. Previously, we showed that WARTS (WTS)/large tumor-suppressor 1 mitotic kinase interacts with the protein/discs-large protein/zonula (PDZ) domain of Omi and promotes its protease activity. We now report that WTS is a substrate for Omi protease activity, thus it is not only a regulator but also a downstream target of this protease. Interaction with Omi PDZ domain is required for WTS to be proteolysed. When caspase-9-deficient mouse embryonic fibroblasts (MEFs) were treated with staurosporine, WTS was proteolysed by activated endogenous Omi without induction of cell death. Therefore, protease activity of Omi and proteolysis of WTS are not necessarily required for cell death. We found that depletion of Omi from HeLa cells results in accelerated cell proliferation despite no significant change in the duration of mitosis. The depletion of WTS showed the same effect on S phase progression. Therefore, WTS proteolytic fragment(s) generated by Omi may act as an inhibitor of G1/S progression. Our data reveal a role for Omi-mediated processing of WTS in negative regulation of cell cycle progression at interphase, suggesting a novel function of Omi other than apoptosis.

Overexpression of manganese superoxide dismutase mRNA may correlate with aggressiveness in gastric and colorectal adenocarcinomas.

The expression or activity of manganese superoxide dismutase (Mn-SOD) is reduced in a variety of malignant tumors and Mn-SOD may act as a new type of tumor suppressor gene. On the other hand, increased expression of Mn-SOD can diminish the cytotoxic effects of several anticancer modalities, including tumor necrosis factor alpha, ionizing radiation, certain chemotherapeutic agents and hyperthermia. Although Mn-SOD expression and its role in various cancers are intensely studied, little is known about its function in gastrointestinal carcinomas. To examine the expression level and significance of Mn-SOD in gastrointestinal carcinomas, Mn-SOD mRNA expression was examined in 53 gastric carcinoma and 38 colorectal carcinoma by reverse transcription-polymerase chain reaction and was compared with those in the corresponding normal mucosal tissues. The tumor/normal (T/N) ratio was calculated and the data were clinicopathologically analyzed. The average T/N ratios of Mn-SOD mRNA expression in gastric and colorectal carcinomas were 2.19 and 3. 72, respectively. Clinicopathologic analyses revealed positive correlation between the Mn-SOD expression level and venous invasion in both gastric and colorectal carcinomas (p<0.05 and p<0.05, respectively). Furthermore, the colorectal carcinoma with lymph node metastasis showed significantly higher Mn-SOD expression than those without it (p<0.05). Our results suggest that Mn-SOD mRNA overexpression can occur in gastric and colorectal carcinomas and may be related to increased aggressiveness.

HLA class I and class II expression of pulmonary adenocarcinoma cells and the influence of interferon gamma.

BACKGROUND: The clinico-biological significance of HLA (both class I antigen and class II one) expressed on tumor cells still remains controversial. METHODS: Tumor cells were freshly separated from 33 surgical specimens of pulmonary adenocarcinoma. The tumor cells were incubated for 24 h in the presence or absence of IFN-gamma (130 International Units/ml). After incubation, the cells were cytocentrifuged onto glass slides and immunostained with either an anti-HLA class I (A, B, C) monoclonal antibody or anti-HLA class II (DR) one. RESULTS: In 22 of 33 cases (66.7%), the HLA class I were individually expressed by more than 60% of tumor cells while so were the HLA class II in 15 (45.4%). No significant correlation was observed between the HLA class I expression and the HLA class II one. The proportion of HLA class I-positive tumor cells correlated with neither the grade of histological differentiation nor the stage of disease. In contrast, the proportion of HLA class II-positive tumor cells correlated with both the grade of histological differentiation and the stage. In most cases, IFN-gamma was found to increase the proportion of class II-positive tumor cells as well as that of class I-positive cells. CONCLUSIONS: The above findings thus suggested that the HLA class II expression might therefore represent a manifestation of cellular differentiation and that IFN-gamma may, as a result, have the potential to differentiate cancer cells.

Failure in resection of multiple pulmonary metastases from colorectal cancer.

BACKGROUND: To clarify whether or not multiple pulmonary metastases from colorectal cancer are contraindicated for a surgical resection, we retrospectively evaluated the influence of the number of pulmonary metastases on both the postthoracotomy survival and the pattern of the first failure. METHODS: From 1981 to 1993, 36 patients underwent a complete resection for pulmonary metastases from colorectal cancer. RESULTS: Of the various factors investigated including gender, primary site, disease-free interval, tumor size, the number of metastases, type of resection, and the history of hepatic metastases, only the number of pulmonary metastases was found to be significantly related to postthoracotomy survival. The rate of disease-free survival at 5 years was 62% for solitary metastasis (n = 17), 35% for two metastases (n = 8), and 0% for four or more metastases (n = 11). The pattern of failure also differed according to the number of pulmonary metastases. In particular, the incidence of local recurrence at the primary site increased with the number of pulmonary metastases (ie, 1 of 17 patients with a solitary metastasis, 3 of 8 with two metastases, and 6 of 11 with four or more metastases). CONCLUSIONS: These results suggest that multiple metastases might indicate the presence of local recurrence at the primary site; therefore, in cases of multiple pulmonary metastases, the primary site should be thoroughly explored.

Long-term survivors with pN2 non-small cell lung cancer after a complete resection with a systematic mediastinal node dissection.

OBJECTIVE: A substantial number of surgical patients with pN2 disease have survived longer than 5 years without any evidence of recurrence, although the surgical indications for those patients remain controversial. The present study was performed in order to clarify the clinical characteristics of the long-term survivors with pN2 disease. METHODS: We retrospectively reviewed the cases of 111 patients with pN2 disease who had undergone a complete resection with a systematic mediastinal lymph node dissection from 1974 through 1991. RESULTS: Of the 111 patients with pN2 disease, 20 survived longer than 5 years after a surgical resection. When both the pre- and post-operative conditions were compared between the long-term survivors and the others, the long-term survivors were characterized by significantly higher proportions of cN0 disease (P = 0.031), pT1 disease (P = 0.004), skip metastasis without hilar node metastasis (P = 0.028), and metastasis of a single mediastinal station (0.044). Of those characteristics, only the likelihood of having cN0 disease could be pre-operatively determined. The survival rate of such a population with cN0-pN2 disease was 34.5% at 5 years and 29.6% at 10 years after a complete resection, respectively. CONCLUSIONS: Pathologic N2 patients with some favorable prognostic factors can survive long-term after a complete resection combined with a systematic mediastinal lymph node dissection. At present, due to the lack of any effective adjuvant therapy, a systematic mediastinal node dissection should be routinely performed even in patients with cN0 disease.

Molecular analysis of a candidate metastasis-associated gene, MTA1: possible interaction with histone deacetylase 1.

We previously identified a novel rat candidate metastasis-associated gene, mta1, based on its differential expression in highly metastatic cells compared to nonmetastatic cells. Furthermore, we showed that overexpression of its human counterpart, MTA1, correlated with the invasiveness or lymph node metastasis of gastric, colorectal and esophageal carcinomas. The aim of this study was to analyze the domains of the MTA1 and investigate the function(s) of this protein. Structural analysis revealed that the MTA1 protein contained a GATA-like zinc-finger domain, a leucine zipper domain, a SANT domain similar to the DNA binding domain of myb-related proteins, a src homology 3-binding domain important in protein-protein interactions, two highly acidic regions characteristic of the acidic activation domains of many transcription factors, and nuclear localization signals. Immunofluorescence staining of COS-7 cells transfected with a myc-epitope-tagged MTA1 expression vector clearly showed nuclear localization of MTA1. Coimmunoprecipitation of myc-tagged MTA1 and FLAG-tagged histone deacetylase 1 (HDAC1), followed by western blot analysis using anti-myc and anti-FLAG monoclonal antibodies showed that MTA1 physically bound with HDAC1 in COS-7 cells. Together with the recent finding that the NURD (nucleosome remodeling and histone deacetylase activities) complex contains an MTA1-related gene product, named MTA2, MTA1 may be another component of this complex and be involved in the alteration of chromatin structure and transcription repression.

[Outpatient chemotherapy with oral UFT (tegafur and uracil) and cisplatin against metastatic non-small cell lung carcinoma--an effective case].

A 64-year-old man underwent a left pneumonectomy for squamous cell carcinoma of the left lung in July 1995. In May 1996, right pelvic bone metastasis occurred and was well controlled by the concurrent chemoradiotherapy; UFT (600 mg/body, day 1-14) and cisplatin (80 mg/m2, day 8) with a total 50.4 Gy of irradiation. In August 1996, left renal metastasis occurred, but regressed after two cycles of combination chemotherapy with UFT and cisplatin. In January 1997, multiple lung metastasis occurred. In accordance with the patient's request, combination chemotherapy was performed at the outpatient clinic. UFT (250 mg/m2) was given orally every day while cisplatin (60-80 mg/m2), fractionated in 3 or 5 days, was intravenously administered at an interval of 4 weeks or longer. The patient has continued the treatment for one year without serious (G3, 4) adverse events. During the treatment, the tumor growth was slow with a repeated cycle of progression and regression. The outpatient chemotherapy using UFT and cisplatin is considered to be useful, especially for the better quality of life of patients.

c-MYC overexpression with loss of Ink4a/Arf transforms bone marrow stromal cells into osteosarcoma accompanied by loss of adipogenesis.

The development of cancer is due to the growth and proliferation of transformed normal cells. Recent evidence suggests that the nature of oncogenic stress and the state of the cell of origin critically affect both tumorigenic activity and tumor histological type. However, this mechanistic relationship in mesenchymal tumors is currently largely unexplored. To clarify these issues, we established a mouse osteosarcoma (OS) model through overexpression of c-MYC in bone marrow stromal cells (BMSCs) derived from Ink4a/Arf (-/-) mice. Single-cell cloning revealed that c-MYC-expressing BMSCs are composed of two distinctly different clones: highly tumorigenic cells, similar to bipotent-committed osteochondral progenitor cells, and low-tumorigenic tripotent cells, similar to mesenchymal stem cells (MSCs). It is noteworthy that both bipotent and tripotent cells were capable of generating histologically similar, lethal OS, suggesting that both committed progenitor cells and MSCs can become OS cells of origin. Shifting mesenchymal differentiation by depleting PPARγ in tripotent MSC-like cells and overexpressing PPARγ in bipotent cells affected cell proliferation and tumorigenic activity. Our findings indicate that differentiation potential has a key role in OS tumorigenic activity, and that the suppression of adipogenic ability is a critical factor for the development of OS.

Suppression of manganese superoxide dismutase augments sensitivity to radiation, hyperthermia and doxorubicin in colon cancer cell lines by inducing apoptosis.

Increased expression of manganese superoxide dismutase (Mn-SOD), one of the mitochondrial enzymes involved in the redox system, has been shown to diminish the cytotoxic effects of several anti-cancer modalities, including tumour necrosis factor-alpha, ionizing radiation, certain chemotherapeutic agents and hyperthermia. We asked if Mn-SOD is a potential target to augment the sensitivity of cancer cells to various anti-cancer treatments and for this we established stable Mn-SOD antisense RNA expressing cell clones from two human colon cancer cell lines, HCT116 (p53 wild-type) and DLD1 (p53 mutant-type). Suppression of Mn-SOD in HCT116 was accompanied by an increased sensitivity to radiation, hyperthermia and doxorubicin, as compared with findings in controls. The mitochondrial permeability transition, as measured by a decrease of the mitochondrial transmembrane potential was more intensely induced by radiation in HCT116 antisense clones than in the control, an event followed by a greater extent of DNA fragmentation. Apoptosis was also induced by hyperthermia more intensely in HCT116 antisense clones than in the control. On the other hand, DLD1 antisense clones did not exhibit any enhancement of sensitivity to any of these treatments. These data support the possibility that inhibition of Mn-SOD activity renders colon cancer cells with wild-type p53 susceptible to apoptosis induced by radiation, hyperthermia and selected anti-cancer drugs. Therefore, we suggest that Mn-SOD could be a target molecule to overcome the resistance to anti-cancer treatments in some colon cancer cells carrying wild-type p53.

The influence of enhanced postoperative inflammation by the intrapleural administration of streptococcal preparation (OK-432) on the prognosis of completely resected non-small-cell lung cancer.

BACKGROUND AND OBJECTIVES: It is not clear whether postoperative inflammation affects the prognosis of malignant disease. METHODS: We retrospectively reviewed the patients with non-small-cell lung cancer who underwent a complete resection at the National Kyushu Cancer Center from 1989 to 1996. For the treatment of prolonged air leakage after a pulmonary lobectomy, 25 patients received an intrapleural injection of OK-432, a lyophilized preparation of the heat- and penicillin-treated Su-strain of the Streptococcus pyogenes group A3. All patients were males who were older than 50 years of age. As a control, we selected 164 male patients who were older than 50 years of age and not given OK-432 during the same period. RESULTS: The administration of OK-432 in most patients was performed on the 4th day after the operation. Pleural drainage could be terminated in a mean of 5.5 days after the intrapleural administration of OK-432. In the control group, the serum C-reactive protein (CRP) level reached a peak on day 4 after the operation and returned to almost a normal level on day 14 after the operation. In the OK-432 group, the peak CRP level, which was significantly higher than that in the control group, was observed on day 7 after the operation and the elevated CRP level was maintained until 28 days after the operation. The mean level of CRP in the OK-432 group was significantly higher than that in the control on days 7, 14, and 28 after the operation. No significant difference was observed in the disease-free survivals between the two groups. CONCLUSIONS: Based on the above findings, postoperative prolonged inflammation does not seem to affect the progression of subclinically residual tumor cells.

Direct influences of pro-inflammatory cytokines (IL-1beta, TNF-alpha, IL-6) on the proliferation and cell-surface antigen expression of cancer cells.

Pro-inflammatory cytokines, e.g. interleukin 1 (IL-1), tumour necrosis factor alpha (TNF-alpha), IL-6 produced by surgical intervention or non-specific immunotherapy may directly affect both the growth and the metastasis of tumour cells. It is therefore important to clarify the direct influence of pro-inflammatory cytokines on tumour cells in order to obtain a better knowledge of anti-tumour therapy. Four human lung cancer cell lines were used. The tumour cells were incubated for 72 h in the presence of various concentrations of IL-1beta, TNF-alpha, or IL-6 and then the proliferative response was assessed by an MTT assay. After 14 days of culture with each pro-inflammatory cytokine, the cell-surface antigen expressions (HLA-class I, HLA-class II, CEA, sialyl Lewis(x)) were assessed by an immunocytochemical staining method. Among the various combinations of tumour cells (PC-9, PC-12, QG-56, QG-95) and cytokines (IL-1beta, TNF-alpha, IL-6), only TNF-alpha significantly exhibited an antiproliferative effect against PC-9 cells. However, various modulations of the cell-surface antigen expression by the cytokines were observed. The HLA-class I antigen expression of PC-9 was augmented by either TNF-alpha or IL-1beta. Furthermore, IL-1beta was able to induce CEA in PC-9, QG-56, and QG-95 cells while TNF-alpha was able to enhance the expression of sialyl Lewis(x)in QG-95 cells. Although the influence of pro-inflammatory cytokines on the growth of tumour cells was only slight, some modulations of the cell-surface antigen expression were notable. The augmentation of HLA-class I expression can thus improve the immunogenicity of tumour cells while the induction of CEA or sialyl Lewis(x)may therefore be associated with the promotion of metastasis.

Intraoperative intrapleural hypotonic cisplatin treatment for carcinomatous pleuritis.

BACKGROUND AND OBJECTIVES: We recently developed a new intraoperative intrapleural hypotonic cisplatin treatment for carcinomatous pleuritis found at thoracotomy in non-small cell lung cancer patients. In the present study, the efficacy and adverse events of this treatment as well as the pharmacokinetics of cisplatin in the blood after the treatment were evaluated. PATIENTS AND METHODS: Twenty-one patients received the treatment for 15 minutes after completing the intrathoracic surgical procedures. The total and free platinum levels in the blood of five patients were then measured. As a control, 29 patients without such treatment were reviewed retrospectively. RESULTS: The survival rates in the treatment and non-treatment groups were similar. The pleural disease free survival of the treated patients was, however, significantly higher than that of the non-treated patients. Such pleural disease as effusion and the growth of the pleural disseminated tumors only appeared in three of the 21 (14%) treated patients while 26 of 29 (90%) non-treated patients had clinically detected pleural disease. The blood platinum levels after the treatment were extremely low and such low levels probably induced no systemic adverse events after the treatment. The only adverse event of this treatment was an increase in the postoperative drainage volume. CONCLUSIONS: These observations seem to suggest that intraoperative intrapleural hypotonic cisplatin treatment for carcinomatous pleuritis found at thoracotomy can, at least, delay the appearance of the pleural disease without any adverse events.

Reduced expression of manganese superoxide dismutase mRNA may correlate with invasiveness in esophageal carcinoma.

Little is known about the expression and antioxidant function of manganese superoxide dismutase (Mn-SOD) in esophageal squamous cell carcinoma. To determine the significance of Mn-SOD in esophageal squamous cell carcinomas, Mn-SOD mRNA expression was examined in 45 esophageal squamous cell carcinomas and the corresponding normal mucosal tissues by reverse transcription-polymerase chain reaction. The tumor/normal (T/N) ratio of 45 patients with esophageal carcinoma was calculated, and the data were clinicopathologically analyzed. The T/N ratio of Mn-SOD mRNA expression was less than 0.5 in 11 (32.4%) of 34 esophageal carcinoma cases without any preoperative treatments, while none of 11 cases who underwent preoperative chemotherapy showed a T/N ratio of <0.5 (p < 0.05). There was an inverse correlation between the Mn-SOD expression level and the degree of venous invasion (p < 0.05) as well as lymphatic invasion (p < 0.05). Furthermore, poorly differentiated squamous cell carcinoma showed significantly lower Mn-SOD mRNA expression levels than well differentiated carcinoma (p < 0.05). Our results suggest that Mn-SOD mRNA was frequently reduced in esophageal carcinoma when compared to the normal mucosa and the reduced expression levels of Mn-SOD mRNA may lead to an accumulation of superoxide radicals in conjunction with the increased invasiveness of esophageal carcinoma.