Hiroki Kusumoto

EXPRESSION OF THE METASTASIS-ASSOCIATED MTA1 PROTEIN AND ITS RELATIONSHIP TO DEACETYLATION OF THE HISTONE H4 IN ESOPHAGEAL SQUAMOUS CELL CARCINOMAS

Metastasis‐associated protein MTA1 and histone deacetylase form a protein complex with histone deacetylase activity that plays an important role in histone deacetylation, alteration of chromatin structure and transcriptional control. The precise role of the MTA1 protein in the malignant progression of human cancers remains unknown, however, especially its overexpression and relationship with histone acetylation/deacetylation in experimental and clinical tumors. The expression levels of MTA1 protein and the acetylation levels of histone H4 were examined in 70 cases of surgically resected esophageal squamous cell carcinomas, using immunohistochemistry. The intensities of immunostaining of MTA1 protein and acetylated histone H4 in carcinoma tissues (Ca) were compared to normal epithelium (N) contained in the same section. Thirty of 70 cases (42.9%) displayed overexpression of MTA1 protein (N < Ca). Cancers overexpressing MTA1 protein invaded deeper into the esophageal wall (p < 0.005) and showed significantly higher degrees of lymph node metastasis (p < 0.01), higher pathological stage, more lymphatic involvement and poorer prognosis (p < 0.05) than the remaining cases. The acetylation levels of histone H4 inversely correlated to the depth of cancer invasion and pathological stage (p < 0.05), and the patients with higher level of histone H4 acetylation had a better prognosis (p < 0.05). Furthermore, immunostaining patterns of MTA1 and acetylated histone H4 were inversely correlated (p < 0.001), demonstrating the relationship of deacetylation of histone H4 in MTA1‐overexpressing carcinomas. In conclusion, the data suggest that the overexpression of MTA1 protein and acetylation level of histone H4 protein, both of which are closely related, might be useful predictors of malignant potential of esophageal squamous cell carcinomas. Thus, strategies involving inhibition of MTA1 function as well as inhibition of histone deacetylation could be novel approaches for the treatment of esophageal squamous cell carcinomas.

Analysis of MT1‐MMP and MMP2 expression in human gastric cancers

Membrane‐type 1 matrix metalloproteinase (MT1‐MMP) is a presumed activator of MMP2, which is one of the major proteinases in tumor cell invasion. In this study, we determined the clinico‐pathologic significance of MT1‐MMP expression in 68 human gastric carcinomas. The tumor‐normal ratio (T/N ratio) of MT1‐MMP expression was determined by reverse transcription‐polymerase chain reaction analysis. To visualize the localization of MT1‐MMP, an immunohistochemical study was performed. In addition, a gelatin zymography was done to examine the activation ratio of MMP2, and a correlation between MT1‐MMP expression and activation of MMP2 was studied. The expression of MT1‐MMP mRNA was higher in tumor tissue than in corresponding normal tissue in most cases. The mean value of the T/N ratio was 4.8. Twenty cases with T/N ≥ 4.8 showed significantly deeper invasion and higher frequency of lymph node metastasis than 48 cases with T/N < 4.8. MT1‐MMP expression was an independent factor influencing both tumor invasion of the gastric wall and lymph node metastasis. Although MT1‐MMP expression was not an independent prognostic factor, the patients with T/N ≥ 4.8 showed a significantly worse prognosis than those with T/N < 4.8. An immunohistochemical study demonstrated that MT1‐MMP expression was mainly recognized in the tumor cells. There was a significant correlation between MT1‐MMP expression and activation of MMP2. Our findings suggest that: 1) the expression of MT1‐MMP may influence prognosis via tumor invasion of the gastric wall and lymph node metastasis, and 2) MT1‐MMP activation of MMP2 may be clinically relevant in gastric carcinoma tumors. Int. J. Cancer 74:316‐321, 1997.

Prophylactic lymph node dissection in patients with advanced gastric cancer promotes increased survival time

Background. There is no consensus of opinion regarding the efficacy of lymph node dissection.

Clinical implications of serum anti-p53 antibodies for patients with gastric carcinoma

Mutations of p53 can lead to the production of anti‐p53 antibodies in sera of cancer patients. Before this study, the value of preoperative serum anti‐p53 antibodies in determining the prognoses of patients with gastric carcinoma had yet to be determined.

5‐fluorouracil and uft‐sensitive gastric carcinoma has a high level of thymidylate synthase

The authors examined the relationship between the level of thymidylate synthase (TS) and the sensitivity to 5‐fluorouracil (5‐FU) and UFT, a combined oral preparation of 1‐(2‐tetrahydrofuryl)‐5‐fluorouracil (tegafur) and uracil in a molar ratio of 1:4. For the studies we used the subrenal capsule (SRC) assay and 15 human gastric cancer tissues. The TS levels were assayed by the ligand‐binding technique, using [6–3H]FdUMP. The relative variation of tumor size (ΔTuS/TuS0) was calculated to be as follows: ΔTuS/TuS0 = [(TuS6 – TuS0/TuS0] X 100 (%), where TuS6 was the tumor size on day 6 and TuS0 on day 0. The chemosensitivity was considered to be positive when ΔTuS/TuS0 in the treated group decreased to below –10%. Decrease in tumor size was marked in case of exposure to UFT (‐19.8 ± 13.0%) (mean ± standard deviation), compared with that to 5‐FU (‐9.0 ± 7.2%), with a statistically significant difference (P < 0.001). The TS level varied from 1.7 to 30.8 pmol/g gastric cancer tissue and the mean was 7.1 ± 7.2 pmol/g tissue. A correlation was noted between the TS level and decrease in size of the tumor exposed to 5‐FU (r = −0.671) or UFT (r = −0.758): gastric cancer tissue with higher level of TS is more sensitive to 5‐FU and UFT than is that with a lower TS level. These findings show that the sensitivity to 5‐FU and UFT of gastric cancer tissue is related to the TS level and that UFT shows promise for the treatment of patients with gastric cancer.

Differences in mutant p53 protein stability and functional activity in teniposide-sensitive and -resistant human leukemic CEM cells

We examined p53 protein stability and DNA damage-induced p53-dependent responses in a human leukemic CEM cell line and two teniposide-resistant sublines, CEM/VM-1 and CEM/VM-1-5 (∼40 and 400-fold resistant to teniposide, respectively). Although all cell lines contain the same p53 mutations at codons 175 (Arg→His) and 248 (Arg→Gln), the constitutive levels of p53 were progressively increased with the resistance of the cells to teniposide. By pulse-chase experiments, we found that the half-lives of mutant p53 protein were approximately 12, 17, and >30 h in CEM, CEM/VM-1, and CEM/VM-1-5 cells, respectively. The prolonged half-lives of p53 in these cells is consistent with the fact that the protein harbors the indicated mutations. Of note, however, is the fact that the increased p53 protein half-lives in the two drug-resistant cell lines corresponds to a proportional decrease in MDM2 protein levels but an increase in p53-MDM2 binding interactions. This suggests that MDM2-mediated p53 degradation may be altered in our leukemic cell lines. The DNA damage-induced p53 response is fully functional in the drug-sensitive CEM cells containing a mutant p53, but this pathway is attenuated in the drug-resistant cells. Specifically, while the mutant p53 was phosphorylated at serine-15 in response to ionizing radiation in all these cell lines, mutant p53 induction in response to teniposide or ionizing radiation and induction of the p53-target genes, p21 and GADD45 only occurred in the drug-sensitive CEM cells. As assessed by MTT cytotoxicity assay, CEM cells were also significantly more sensitive to ionizing radiation, compared to the drug-resistant cell lines, and this correlated with p53 induction. Collectively, these results suggest that changes in constitutive mutant p53 protein levels, p53-MDM2 binding interactions, and altered regulation of the DNA damage-inducible p53-dependent pathway may play a role in drug- and radiation-responsiveness in these cells.

Sodium Succinate Enhances the Colorimetric Reaction of the in vitro Chemosensitivity Test: MTT Assay

We compared the colorimetric reactions between the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl 2H-tetrazolium bromide (MTT) assay and the succinate dehydrogenase inhibition (SDI) test, in order to evaluate the usefulness of the SDI test for in vitro chemosensitivity testing. The addition of sodium succinate enhanced the colorimetric absorbance at 565 nm in the MTT assay in a dose- and a time-dependent manner, in mouse sarcoma-180 (S-180) cells. At 10 microM of sodium succinate, a dose used in the SDI test, the absorbance of the MTT assay increased by about 2.5-fold in the S-180 cells and in 10 human tumor tissues. The absorbance in the SDI test correlated well with the viable cell number of S-180 cells (r = 0.9993). These results show that the SDI test, using MTT as a tetrazolium salt, has a higher sensitivity for predicting cell viability, compared to the MTT assay.

Comparison between Succinate Dehydrogenase Inhibition Test and Subrenal Capsule Assay for Chemosensitivity Testing

The chemosensitivity result of the succinate dehydrogenase inhibition (SDI) test was compared with that of the subrenal capsule (SRC) assay in 23 human tumor tissues exposed to adriamycin (ADM), mitomycin C (MMC), cisplatin (DDP) and 5-fluorouracil (5-FU). The chemosensitivity was considered as positive when the succinate dehydrogenase (SD) activity of the drug-exposed cells was decreased to below 50% of that of control cells on day 3 in the SDI test, and the tumor size on day 6 was decreased to below -10% of that on day 0 in the SRC assay. Correlation rates between the decrease of SD activity in the SDI test and the decrease of tumor size in the SRC assay, using 23 evaluable cases in both assays, were r = 0.717 for ADM, r = 0.699 for MMC, r = 0.796 for DDP and r = 0.735 for 5-FU. The correlations of the chemosensitivity results were 73.9% for ADM, 73.9% for MMC, 82.6% for DDP and 60.9% for 5-FU. A positive correlation was noted between the in vitro and in vivo chemosensitivity results. This SDI test can serve as an effective tool for chemosensitivity testing.

Postoperative chemotherapy for patients with advanced gastric cancer

The relationship between postoperative chemotherapy and survival time after gastric resection in patients with advanced gastric cancer was examined by retrospectively reviewing data on 916 patients treated in our clinics between 1965 and 1985. Of these patients, 738 were treated postoperatively with antitumor drugs. Postoperative chemotherapy was more often prescribed for those in the advanced stages of malignancy. Univariate analysis revealed that the survival time of patients given postoperative chemotherapy was shorter than for those not receiving chemotherapy, but there was no statistical significance. Multivariate analysis using the Cox regression analysis adjusted for sex, age, and other covariants indicated that operative curability, liver metastasis, serosal invasion, lymph node metastasis, peritoneal dissemination, and tumor size were the important prognostic factors. There was no correlation with postoperative chemotherapy. Our findings rule out any relationship between postoperative chemotherapy and length of survival time for patients with advanced gastric cancer undergoing gastric resection.

Colorectal carcinoma in vitro is more sensitive to 1-hexylcarbamoyl-5-fluorouracil compared with six other antitumor drugs: carboquone, Adriamycin, mitomycin C, aclacinomycin A, cisplatin, 5-fluorouracil.

The sensitivity to 1-hexylcarbamoyl-5-fluorouracil (HCFU) of 25 colorectal cancer tissues was compared with that of six antitumor drugs: carboquone (CQ), Adriamycin® (ADM), mitomycin C (MMC), aclacinomycin A (ACR), cisplatin (DDP), and 5-fluorouracil (5-FU), using thein vitro succinate dehydrogenase inhibition (SDI) test. Chemosensitivity was determined to be positive when the succinate dehydrogenase (SD) activity of the drug-exposed cells, at ten times the peak plasma concentration, was decreased to below 50 percent of that of control cells on day 3 of exposure. Decrease in SD activity was remark-able in cases of exposure to HCFU, compared with six other drugs. The sensitivity rates were 32 percent for CQ, 40 percent for ADM, 24 percent for MMC, 28 percent for ACR, 32 percent for DDP, 16 percent for 5-FU, and 68 percent for HCFU. The sensitivity rate for at least one of the six drugs (CQ, ADM, MMC, ACR, DDP, and 5-FU) was 52 percent, but was 80 percent when HCFU was taken into account. Since colorectal cancer tissues are resistant to various antitumor drugs, the chemosensitivity test of HCFU should aid in determining the effects of a particular drug for an individual patient.