The influence of stromal cells on the MTT assay (I)—In vitro chemosensitivity of the tumor and stromal cells to mitomycin C—

To clarify the influence of stromal cells on the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide assay (MTT assay), a gastric carcinoma cell line (KATO-III) and a human fibroblast cell line (IMR-90) were subjected to a colorimetric assay, in which the chemosensitivity to mitomycin C was assessed in different mixtures of the cell lines. KATO-III was found to be highly sensitive to mitomycin C at 10 μg/ml, whereas IMR-90 was insensitive to mitomycin C at the same concentration. When the mixtures of these two cell lines were tested by the assay, a mixture of more than 25 per cent stromal cells reduced the sensitivity of KATO-III to mitomycin C. This suggested that the stromal cells in fresh surgical specimens might reduce the apparent sensitivity of the tumor cells.     

The influence of stromal cells on the MTT assay (II)--Study on the nude mouse system

A fresh surgical specimen from colon carcinoma was tested by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide assay (MTT assay) and transplanted into nude mice. After 5 transfers in male BALB/c nude mice, the xenograft was then tested again by the MTT assay. It was found that the in vivo chemosensitivity pattern of the xenograft was essentially identical to that of the in vitro fresh surgical specimen, whereas the sensitivity of the xenograft was increased. To exclude the stromal cells from the nude mouse, anti-BALB/c serum was added to the primarily cultured colon carcinoma xenograft, and its chemosensitivity to mitomycin C (MMC) assessed. Although the sensitivity of the serum-treated group to MMC was slightly higher than that of the untreated group, the dose-response curves of the tumor cells to MMC were similar to each other. Thus, the chemosensitivity pattern of tumor cells seemed to be stable with or without normal cells, although the sensitivity itself was reduced by the presence of normal cells.     

The influence of stromal cells on the MTT assay (II)—Study on the nude mouse system—

A fresh surgical specimen from colon carcinoma was tested by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide assay (MTT assay) and transplanted into nude mice. After 5 transfers in male BALB/c nude mice, the xenograft was then tested again by the MTT assay. It was found that thein vivo chemosensitivity pattern of the xenograft was essentially identical to that of thein vitro fresh surgical specimen, whereas the sensitivity of the xenograft was increased. To exclude the stromal cells from the nude mouse, anti-BALB/c serum was added to the primarily cultured colon carcinoma xenograft, and its chemosensitivity to mitomycin C (MMC) assessed. Although the sensitivity of the serum-treated group to MMC was slightly higher than that of the untreated group, the dose-response curves of the tumor cells to MMC were similar to each other. Thus, the chemosensitivitypattern of tumor cells seemed to be stable with or without normal cells, although thesensitivity itself was reduced by the presence of normal cells.     

In vitro chemosensitivity tests on human tumor xenografts by clonogenic assay: the combined use of mitomycin C with alpha-interferon or gamma-interferon

Using the human tumor clonogenic assay technique, the effects of Mitomycin C plus either alpha-interferon or gamma-interferon were studied against five human tumor xenografts serially transplanted into nude mice (three gastric and two colon cancers). When the survival fraction found with the drug combination was smaller than the multiplication of survival fractions with either drug alone, the combined effect was defined as synergism. Similarly, antagonistic effect was defined when the survival fraction of drug combination was larger than the larger one observed in either interferon or Mitomycin C alone. Four out of five human tumor xenografts (three gastric and one colon cancers) showed synergistic effects in combination of alpha-interferon with Mitomycin C. Though two gastric cancer xenografts exhibited synergistic effects in combination of gamma-interferon with Mitomycin C, antagonistic effects, which was not found in combination of alpha-interferon with Mitomycin C, were observed in one gastric cancer and one colon cancer xenografts.     

In vitro chemosensitivity tests on human tumor xenografts by clonogenic assay: the combined use of mitomycin C with α-interferon or γ-interferon

Using the human tumor clonogenic assay technique, the effects of Mitomycin C plus either α-interferon or γ-interferon were studied against five human tumor xenografts serially transplanted into nude mice (three gastric and two colon cancers). When the survival fraction found with the drug combination was smaller than the multiplication of survival fractions with either drug alone, the combined effect was defined as synergism. Similarly, antagonistic effect was defined when the survival fraction of drug combination was larger than the larger one observed in either interferon or Mitomycin C alone. Four out of five human tumor xenografts (three gastric and one colon cancers) showed synergistic effects in combination of α-interferon with Mitomycin C. Though two gastric cancer xenografts exhibited synergistic effects in combination of γ-interferon with Mitomycin C, antagonistic effects, which was not found in combination of α-interferon with Mitomycin C, were observed in one gastric cancer and one colon cancer xenografts.     

Hypoxia enhances the lethality of mitomycin C and carboquone against human malignant tumor cells in vitro

Solid tumors contain hypoxic cells which are relatively resistant to antineoplastic activity of radiation or to chemotherapeutic agents. We determined the increase in antineoplastic activities of mitomycin C (MMC) and carboquone (CQ) against human tumor cells under conditions of in vitro hypoxia, with the following results. (a) The HeLa cells had been exposed to various concentrations of MMC or CQ for 2 h under normally aerated (about 20%) or hypoxic conditions (5.0 and 0%) and were maintained under normally aerated conditions for 3 days. The succinate dehydrogenase activity was assayed, using the succinate dehydrogenase inhibition (SDI) test. Treatment with MMC or CQ reduced the SD activity, compared to findings in the control cells, under hypoxic conditions. (b) The cells were exposed to the drug, MMC or CQ, under normally aerated or anoxic conditions, for 15-45 min and the colonies counted on day 14. Treatment with 0.45 microM of MMC for 45 min inhibited the clonogenicity of HeLa cells by 25.4% of findings in the control cells under anoxia. Treatment with 0.03 microM of CQ for 30 min inhibited the clonogenicity by 41.5%. (c) The sensitivity of 14 human tumor tissues (6 gastric and 8 colorectal cancers) to MMC or CQ under hypoxic conditions was determined, using the SDI test. MMC and CQ showed hypoxic enhancement. Therefore, MMC and CQ have definite positive effects on hypoxic cells, and hence a higher therapeutic ratio for treating clinical solid tumors.     

肿瘤体外药敏试验三磷酸腺苷生物发光法在肝癌化疗中的应用

目的：探讨肿瘤体外药敏试验三磷酸腺苷生物发光法（ATP－TCA）在肝癌综合治疗中的应用。方法：取36例肝癌手术、活检和穿刺标本行ATP－TCA。结果：ATP－TCA的可评估率为94．44％；10种化疗药物的敏感率分别为：5－氟脲嘧啶（5－Fu）8．33％、诺消灵（MIT)8．33％、顺铂（CDDP）11．11％、草酸铂（OXA）13．89％、足叶已甙（VP－16）19．44％、丝裂霉素（MMC）25．00％、表阿霉素（EPI）27．78％、开普拓（CPT）44．44％、健择（GEM）47．22％、泰素（TAX）63．89％。结论：ATP－TCA法可用于肝癌化疗中化疗药物的筛选。     

生物可降解材料聚β-羟基丁酯对兔骨髓基质细胞生物学特性的影响

目的：检测生物可降解材料聚β-羟基丁酯(PHB)对兔骨髓基质细胞(BMSC)形态、生长、附着及增殖的影响。方法：将家兔BMSC进行体外培养，以诱导剂(β-甘油磷酸钠，地塞米松，L-抗坏血酸)使其向成骨细胞分化，行碱性磷酸酶染色并计算阳性率，将PHB与成骨细胞样细胞进行体外复合培养，用倒置显微镜及扫描电镜观察PHB对BMSC的形态、生长、附着及增殖的影响。结果：在诱导剂的作用下BMSC向成骨细胞分化，碱性磷酸酶染色呈阳性，其阳性率随培养时间延长而增加。PHB与成骨细胞样细胞进行体外复合培养后显示PHB对BMSC的形态、生长、附着及增殖均无不良影响。结论：PHB具有良好的生物相容性，可望与骨髓复合移植修复骨缺损。     

体外化疗药敏试验系统联合流式细胞仪在胃肠肿瘤化疗中的应用

目的：探讨体外化疗药敏试验系统（ATP－TCA系统）联合流式细胞仪（FCM）在胃肠肿瘤化疗中的应用。方法：取36例胃癌、结直肠癌的手术标本行ATP－TCA试验和FCM P170检测，并分析两结果之间的关系。结果：ATP－TCA的可评估率为88．1％，10种化疗药物的敏感率分别为：氟尿嘧啶15．6％，顺铂18．8％，足叶乙甙21．9％，草酸铂25．0％，丝裂霉素31．3％，表阿霉素37．5％，诺消灵40．6％，开普拓43．8％，健择53．1％，泰素56．3％，P170检测阳性率为43．8％，丝裂霉素，表阿霉素，诺消灵的敏感率在P170阴性表达时高，开普拓，健择，泰素在P170阳性和阴性表达时的敏感率均高，且差异无显性意义（P＞0．05），结论：ATP－TCA法联合FCM可较好地用于胃肠肿瘤的化疗药物的筛选，开普拓，健择，泰素可用于P170阳性表达的患。     

Effect of oxygen tension on the antitumor activity of mitomycin C assayed by human tumor clonogenic assay

The effects of oxygen tensions on the antitumor activity of mitomycin-C (MMC) were surveyed using the human tumor clonogenic assay technique. Six human tumor xenografts (4 gastric cancers, and 2 colon cancers) were used in this study. Tumor cells were continuously exposed to MMC during the experimental period at various oxygen tensions, such as 2 per cent, which is considered to be hypoxic oxygen tension, 5 per cent which is considered as the physiological oxygen tension, and 20 per cent which is the conventional in vitro culture condition. The antitumor activities of MMC on the 6 human tumor xenografts increased when the oxygen tension was lowered from 20 per cent to 5 per cent. However, no further increases of the antitumor activities of MMC were observed by lowering the oxygen tension from 5 to 2 per cent. Additionally, the in vitro antitumor activities of MMC at various oxygen tensions were compared with the in vivo chemosensitivities evaluated in nude mice. In five of the 6 human tumor xenografts, in vitro chemosensitivities assayed at 2 or 5 per cent oxygen tension were concordant with in vivo chemosensitivities, although in vitro chemosensitivities assayed at 20 per cent were concordant with in vivo chemosensitivities in 3 of the 6 tested tumors.     

Effect of oxygen tension on the antitumor activity of mitomycin C assayed by human tumor clonogenic assay

The effects of oxygen tensions on the antitumor activity of mitomycin-C (MMC) were surveyed using the human tumor clonogenic assay technique. Six human tumor xenografts (4 gastric cancers, and 2 colon cancers) were used in this study. Tumor cells were continuously exposed to MMC during the experimental period at various oxygen tensions, such as 2 per cent, which is considered to be hypoxic oxygen tension, 5 per cent which is considered as the physiological oxygen tension, and 20 per cent which is the conventionalin vitro culture condition. The antitumor activities of MMC on the 6 human tumor xenografts increased when the oxygen tension was lowered from 20 per cent to 5 per cent. However, no further increases of the antitumor activities of MMC were observed by lowering the oxygen tension from 5 to 2 per cent. Additionally, thein vitro antitumor activities of MMC at various oxygen tensions were compared with thein vivo chemosensitivities evaluated in nude mice. In five of the 6 human tumor xenografts,in vitro chemosensitivities assayed at 2 or 5 per cent oxygen tension were concordant within vivo chemosensitivities, althoughin vitro chemosensitivities assayed at 20 per cent were concordant within vivo chemosensitivities in 3 of the 6 tested tumors.     

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目的 探讨体外化疗药物敏感试验系统（ATP-TGA系统）在消化道恶性实体肿瘤的应用及体会。方法 取肝癌，胃癌，结肠癌，直肠癌共42例的手术，活检和穿刺标本应用体外药敏试验ATP-TCA法。结果 ATP-TCA的可评估率为88.10%,10种化疗药物的敏感率分别为：氟脲嘧啶11.11%，顺铂13.89%。足叶乙甙19.44%，草酸铂22.22%。诺消灵25.00%。丝裂霉素27.78%，表阿霉素38.89%。开普拓44.45%。健择52.78%。泰素62.16%。结论 ATP-TCA法可用于消化系恶性实体肿瘤化疗中的化疗药物的筛选。     

Analysis of the chemosensitivity test by human tumor clonogenic assay and subrenal capsule assay

The antitumor activities of several chemotherapeutic agents were tested in the human tumor clonogenic assay (HTCA) and in subrenal capsule assay (SRCA). The results obtained in these two assays and clinical response were compared. 1. Colony growth was observed in 43 of 73 tumors (58.9%), and the adequate colony growth to evaluate the response of therapeutic groups was obtained in 21 tumors (28.8%). 2. Control growth adequate to meet evaluable assay criteria was obtained in 38 of 43 tumors (88.4%). 3. With activity criteria set at a decrease of greater than or equal to 50% in tumor colony-forming units for the HTCA and a change in tumor size less than or equal to -1.0 dmm for the SRCA, 24 of 98 drugs tested were active in HTCA (24.5%), and 50 of 160 drugs were active in SRCA (31.2%). 4. A total of 21 tumors was tested in both HTCA and SRCA and the tumor response was compared in 12 tumors treated with 39 drugs. Correlation of tumor responses between the two assays was 71.8%. 5. A total of 8 HTCA-clinical correlations and 5 SRCA-clinical correlations were possible. Overall predictive accuracy was 87.5% in the HTCA and 80.0% in the SRCA, respectively.     

Comparative studies on in vitro human tumor clonogenic assay (HTCA) and in vivo nude mouse-isotope assay (NM-IA)

Comparative studies between the in vitro human tumor clonogenic assay (HTCA) and nude mouse-isotope assay (NM-IA), in which the final evaluation was made with 3H-thymidine incorporations of tumor cells transplanted into nude mice, were performed simultaneously on 60 fresh human tumors. Tissues used included 27 gastric cancers, 10 breast cancers, 7 colorectal cancers, 4 gallbladder cancers, 4 sarcomas, 3 lymphomas, and 5 other tumors. Mitomycin C (MMC), 5-fluorouracil (5-FU), cyclophosphamide (CPM) and adriamycin (ADM) were tested. The overall evaluable rate was 66.7 per cent in HTCA and 83.3 per cent in NM-IA, respectively. When the per cent survival in HTCA and the per cent inhibition in NM-IA less than 50 per cent was defined as drug sensitive, the drug sensitive rates of MMC, 5-FU, CPM, and ADM were 23.1, 16.7, 11.8, and 27.8 per cent in HTCA and 17.6, 18.4, 22.4, and 25.5 per cent in NM-IA, respectively. Although statistically significant correlations between the results of HTCA and those of NM-IA were obtained for MMC and ADM, no correlation was observed for 5-FU and CPM. The overall predictive accuracy rate of clinical response was 83.3 per cent (true positive rate 50 per cent and true negative rate 92.9 per cent) in HTCA and 76.0 per cent (true positive rate 37.5 per cent and true negative rate 93.8 per cent) in NM-IA, respectively.     

Clinical characteristics influence in vitro action of 1,25-dihydroxyvitamin D(3) in human marrow stromal cells

Vitamin D is important for bone health, with low vitamin D levels being associated with skeletal fragility and fractures. Among its other biological activities, 1,25-dihydroxyvitamin D (1,25(OH)₂D), stimulates the in vitro differentiation of human marrow stromal cells (hMSCs) to osteoblasts, which can be monitored by increases in alkaline phosphatase enzyme activity or osteocalcin gene expression. In this study, we tested the hypotheses that age and clinical attributes of subjects influence in vitro responsiveness of hMSCs to 1,25(OH)₂D₃. In a cohort of subjects whose hMSCs were isolated from bone marrow discarded during hip replacement surgery for osteoarthritis, there were significant inverse correlations with age for bone mineral density, renal function, body mass index, fat mass index, and lean mass index (n = 36–53). There were significant correlations with serum 25(OH)D for serum parathyroid hormone (PTH), body mass index, fat mass index, and lean mass index (n = 47–50). In vivo–in vitro correlation analyses indicated that there were significantly greater in vitro effects of 1,25(OH)₂D₃ to stimulate osteoblast differentiation in hMSCs obtained from subjects who were younger than 65 years of age, or who had serum 25(OH)D ≤ 20 ng/mL, elevated serum PTH, or better renal function, assessed by estimated glomerular filtration rate. The greater in vitro stimulation of osteoblast differentiation by 1,25(OH)₂D₃ in hMSCs from vitamin D-deficient subjects suggests that vitamin D replenishment may lead to more vigorous bone formation in subjects at risk. © 2012 American Society for Bone and Mineral Research.     

MTT测定恶性肿瘤细胞对化疗药物的敏感性

目的 探讨３－（４．５）－双甲基－２－噻唑－（２．５）－二苯基溴化四氮唑蓝（ＭＴＴ）法测定恶性肿瘤细胞对化疗药物的敏感性。方法 采用改良的ＭＴＴ法检测了１８９例恶性肿瘤标本对９种化疗药物的体外敏感性。结果 ５１例胃癌细胞对化疗药物的敏感性,由高到低依次为丝裂霉素（ＭＭＣ）,５－氟脲嘧啶（５－Ｆｕ）,顺铂（ＤＤＰ）,阿霉素（ＡＤＭ）,阿糖胞苷（Ａｒａ－Ｃ）,平阳霉素（ＰＹＭ）,甲氨喋呤（ＭＴＸ）,足     

c-kit and PDGFRA mutations in extragastrointestinal stromal tumor (gastrointestinal stromal tumor of the soft tissue)

Extragastrointestinal stromal tumor (EGIST) is a unique tumor that occurs outside the gastrointestinal tract. EGIST shows a c-kit expression and histologic appearance similar to those of gastrointestinal stromal tumor (GIST). Most GISTs have gain-of-functional mutation of the c-kit gene, and some have mutation of the platelet-derived growth factor receptor-alpha (PDGFRA) gene. However, the frequency of mutation of those genes in EGISTs remains unclear. We examined the clinicopathologic features, prognostic factors, and c-kit and PDGFRA mutation in 39 cases of EGIST. Tumors with high mitotic counts (>or=5/50 high power fields) or a high Ki-67 labeling index (>or=10%) were significantly correlated with worse prognoses. The c-kit mutation was found in the juxtamembrane domain (exon 11) and the extracellular domain (exon 9) in 12 of 29 cases (41.4%) and 2 of 29 cases (6.9%), respectively. The PDGFRA gene mutation was found at the juxtamembrane domain (exon 12) and the tyrosine kinase domain (exon 18) in one case each. The pattern of kit and PDGFRA mutation in EGIST was essentially similar to that in GIST. Our results suggest that the c-kit and PDGFRA mutations play an important role in the tumorigenesis of EGIST. High mitotic counts and a high Ki-67 labeling index may be useful for predicting the aggressive biologic behavior in EGIST. Furthermore, STI-571, targeting c-kit and PDGFR tyrosine kinase, seems to be a possible therapeutic strategy for EGISTs, especially advanced cases.     

Sequential intravesical chemotherapy with mitomycin C and adriamycin for superficial bladder tumor (preliminary report)

Intravesical chemotherapy with sequential instillation of mitomycin C and adriamycin was carried out on 19 patients with superficial bladder cancer (Ta, T1 and Tis). Twenty milligram of mitomycin C on day 1 and 40 mg of adriamycin on day 2 were instilled into the bladder, this treatment being repeated weekly for 5 consecutive weeks if there were no serious side effects. The following results were obtained. Of 16 evaluable patients, 10 patients (63%) achieved complete response and 2 patients achieved partial response, 4 patients showing no response. In patients with high grade tumor, especially with carcinoma in situ, a high CR rate (6/7 or 86%) was achieved. Chemical cystitis occurred in 10 out of 19 (53%) patients. In 5 of the 6 patients who suffered from severe cystitis symptoms, treatment was discontinued. However, the symptoms resolved within 4 weeks in all patients.     

Gastrointestinal stromal tumor (gist) of the duodenum

This is a report of a rare gastrointestinal stromal tumor of the duodenum in a 75 years old man who presented with recurrent episodes of intestinal obstruction and melena. The patient underwent successful Whipple's procedure.     

Effects of intraperitoneal mitomycin C adsorbed on activated carbon on adhesion formation and mesothelial cells in vitro.

OBJECTIVE: To investigate the incidence of adhesions after intraperitoneal instillation of mitomycin C adsorbed on activated carbon (MMC-CH). DESIGN: Animal and laboratory studies. SETTING: University hospital, Germany. ANIMALS: 90 Sprague-Dawley rats. INTERVENTIONS: Laparotomy, small bowel anastomosis, and intraperitoneal instillation of saline (controls, n = 27), activated carbon alone (n = 24) or MMC-CH (n = 26). Cultures of monolayers of human mesothelial cells. MAIN OUTCOME MEASURES: Measurements of adhesions by planimetry. Toxicity of mitomycin C alone and charcoal alone in mesothelial cell monolayers as reflected by cell proliferation and measurement of lactate dehydrogenase activity. Concentrations of plasminogen activator (tPA) and plasminogen activator inhibitor 1 (PAI-1) as measures of the fibrinolytic activity of mesothelial cells. RESULTS: Both activated carbon and MMC-CH caused a significant increase of adhesion formation in rats. Activated carbon also reduced the fibrinolytic activity of mesothelial cells, and mitomycin C caused concentration-dependent cytotoxicity in vitro. CONCLUSIONS: Activated carbon combined with high concentrations of mitomycin C may cause intraperitoneal infective complications by increasing the rate of adhesion formation and reducing the fibrinolytic activity of mesothelial cells. We recommend a new absorbable carrier for intraperitoneal chemotherapy.