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Background: Protein kinase C (PKC) is a family of enzymes that function in processes relevant to carcinogenesis, tumor cell metastasis, and apoptosis. Safingol, an optical isomer (the L-threo enantiomer) of dihydrosphingosine, is a specific inhibitor of PKC and might represent a novel agent for anticancer therapy. Preclinical animal studies show that safingol alone has a minimal effect on tumor cell growth, but combining this compound with conventional chemotherapy agents dramatically potentiates their antitumor effects. It has been suggested that many chemotherapeutic agents exert their antitumor effects by inducing apoptosis. Purpose: We wanted to determine the extent to which safingol, alone or in combination with a standard chemotherapeutic agent (mitomycin C [MMC]), would promote apoptosis in gastric cancer cells in vitro. Furthermore, we investigated whether the induction of apoptosis in the treated cells was affected by their p53 tumor suppressor status or their drug-resistance status. Methods: SK-GT-5 (p53-deficient and MMC-resistant) and MKN-74 (p53 wild-type and MMC-sensitive) gastric cancer cells were exposed to either no drug, safingol (50 [micro]M) alone, MMC (5 [micro]g/mL) alone, or a combination of safingol (50 [micro]M) and MMC (5 [micro]g/mL). In some experiments, cells were exposed simultaneously to safingol and the PKC activator, 3-phorbol 12-myristate 13-acetate (PMA), prior to treatment with MMC. Apoptosis was measured by two methods: 1) quantitative fluorescence microscopy of nuclear chromatin condensation in cells stained with the dye, bisbenzamide trihydrochloride (Hoechst-33258), and 2) terminal deoxynucleotidyl transferase (TdT) labeling of the 3'-OH ends of DNA fragments produced in apoptotic cells. Results: As determined by quantitative fluorescence microscopy, exposure of SK-GT-5 cells to safingol alone induced apoptosis in 2% [+ or -] 1% (mean [+ or -] SD) of the cells, and MMC alone increased that level to 18% [+ or -] 1%. However, the combination of safingol and MMC induced apoptosis in 39% [+ or -] 1% of the cells (P

Background. Adenocarcinoma of the proximal stomach is now the most rapidly rising cancer among men in the United States. The development of metastases is the major cause of morbidity and mortality for this aggressive disease. The mechanisms by which tumor cells invade the basement membrane are unknown for this disease. We have identified and established 5 invasive and noninvasive adenocarcinoma cell lines arising from the proximal stomach, which can be used to examine the mechanisms involved in tumor cell invasion. Methods. The expression of factors associated with tumor cell attachment, proteolysis, and inhibition of proteolysis was determined by reverse transcription of mRNA to cDNA and subsequent amplification by the polymerase chain reaction. In addition, cells were examined for morphologic changes by scanning electron microscopy. Results. Invasive proximal gastric cancer cells express the 72-kD form of collagenase type IV, whereas the noninvasive cells do not. Other factors examined (including the laminin receptor, cathepsin B, cathepsin L, urokinase-type plasminogen activator, and tissue inhibitor metalloproteinases) are expressed by both invasive and noninvasive gastric cancer cells, whereas collagenase type IV 92-kD form is not expressed by any of the cells examined. In addition, scanning electron microscopy revealed that all the invasive cell lines exhibit long cytoplasmic extensions. The noninvasive cells express short cytoplasmic projections and are rounder than the invasive proximal gastric cancer cell lines. Conclusions. There are distinct phenotypic differences between invasive and noninvasive proximal gastric cancer cell lines both at the level of expression of mRNA for collagenase Type IV 72-kD and at the level of scanning electron microscopy with the expression of cytoplasmic projections. Clinical outcome may be associated with these phenotypic differences.

Background. Chemotherapy has had little impact on the natural history of soft tissue sarcoma, and often is associated with serious toxicity. Edatrexate, an investigational antifolate, is active in patients with lung cancer, and has cytotoxic activity in human sarcoma cell lines. Methods. Edatrexate was administered to 36 patients with measurable, advanced soft tissue sarcoma who had not previously received chemotherapy. The drug was given weekly for 5 weeks, then every other week. The initial dose, 80 Mg/[m.sup.2], was escalated by 10 Mg/[m.sup.2] every 2 weeks in the absence of toxicity. Eleven patients had leiomyosarcoma, 7 had malignant fibrous histiocytoma (MFH), and 5 had liposarcoma; the remainder of cell types included hemangiopericytoma (4), angiosarcoma (3), synovial (2), spindle cell (2), extraosseous chondrosarcoma (1), and fibrosarcoma (1). Results. Thirty-five patients are evaluable. Partial response (PR) was seen in five of the seven patients with MFH; no other major responses occurred. Overall, the response frequency was 14% (two-sided 95% confidence interval, 3% to 26%). Median duration of PR was 6 months (range, 4-18 months). One patient had a minor tumor regression, and six had stable disease. Myelosuppression was generally mild; only three patients had grade 3 hematologic toxicity. Modification of dose or schedule was required in 50% of patients for mucositis. Fatigue was a common toxicity, seen in 66% of patients, but was tolerable in the majority. A rash was seen in 46% of patients; one patient had hepatic toxicity. Conclusions. Overall, the activity of edatrexate in this study, dominated by patients with either visceral or vascular sarcoma, was poor. However, the responses observed in patients with metastatic MFH suggests that further evaluation of edatrexate in patients with soft tissue sarcoma is warranted.

Background. Gastric carcinoma is one of the most common malignancies worldwide, with an overall survival of about 10%. Improvement in therapy awaits better understanding of the biologic behavior of this tumor. Establishment of cell lines permits detailed analysis of the biology of gastric cancer. The authors report on the establishment and characterization of five cell lines arising from primary proximal gastric and distal esophageal adenocarcinomas. Methods. Cultures of epithelial cells from adenocarcinomas of the proximal stomach or adenocarcinoma of the lower esophagus were established. Gastric cancer cell lines were analyzed for doubling times, anchorage-independent growth, tumorigenic and metastatic potential in nu/nu mice, expression of keratin proteins by indirect immunofluorescence, invasive potential in a Boyden Chamber, and growth factor production by reverse transcription of mRNA in cDNA and subsequent amplification by the polymerase chain reaction. Results. Five cell lines were derived from primary gastric adenocarcinomas of the proximal stomach and from Barrett esophagus. All five cell lines were tumorigenic but not metastatic in vivo. None were capable of anchorage independent growth in vitro. Two lines were highly invasive in the Boyden chamber assay, whereas two lines were minimally or noninvasive. All five cell lines expressed RNA transcripts specific for the growth factors TGF[Beta]1, TGF[Beta]2, TGF[Beta]3, TGF[Alpha], and platelet-derived growth factor A, whereas subsets of cell lines expressed transcripts for aFGF, bFGF, FGF-5, Hst, and platelet-derived growth factor B. Conclusions. Five cell lines derived from primary gastric-esophageal adenocarcinomas were established in tissue culture. These cell lines show differences in morphologic features, growth potential, and invasiveness. These newly established gastric cancer cell lines should prove useful for a wide range of studies attempting to decipher the biology of proximal gastric adenocarcinoma.

Background: Gastric adenocarcinoma is a common neoplasm worldwide. Patients with completely resected disease often have locoregional recurrence, and adjuvant chemotherapy has failed to reduce the common occurrence of metastases. Protein kinase C (PKC) is thought to be important in tumor cell invasion, but its relationship to gastric cancer cell invasion, and thus metastases, remains unexplored. We recently identified and established invasive and noninvasive human gastric adenocarcinoma cell lines, which can now be used to test agents for inhibition of tumor cell invasion by inhibition of PKC activity. Purpose: The objectives were (a) to test threo-dihydrosphingosine (SPC100221), a specific inhibitor of PKC at its regulatory site, and staurosporine, a potent but nonspecific inhibitor of PKC at its catalytic site, for their effects on gastric cancer cell invasion in vitro and (b) to determine whether the expression of PKC isoforms can distinguish invasive from noninvasive gastric cancer cells. Methods: Gastric cancer cell invasion through Matrigel-coated Nuclepore filters in the Boyden chamber assay was analyzed in the presence of graded concentrations of SPC100221 and staurosporine. The invasive SK-GT-1 and SK-GT-5 cell lines and the noninvasive SK-GT-2 and SK-GT-4 cell lines were used. PKC isoform expression was determined by reverse transcription of messenger RNAs to complementary DNA and subsequent amplification by the polymerase chain reaction. Results: The effects of staurosporine and SPC100221 on tumor cell invasion were tested at drug concentrations that did not inhibit cell proliferation, as evidenced by [H.sup.3]thymidine uptake. Staurosporine and SPC100221 at subtoxic doses inhibited human gastric cancer cell invasion by 50% at 5 x [10.sup.-9] M and 2 x [10.sup.-7] M, respectively. The expression of PKC[beta] was observed in the invasive but not the noninvasive gastric cancer cells. Both types of cells, however, expressed the PKC[alpha] and PKC[gamma] isoforms. Conclusions: Gastric cancer cell invasion can be inhibited by PKC inhibitors, and expression of PKC[beta] may be a marker of invasiveness in gastric cancer. Implications: PKC appears to represent a new target for inhibition of gastric cancer cell invasion, and SPC100221, in view of its PKC specificity, may provide a model for future drug development in this area. Moreover, PKC[beta] may have a fundamental role in the development of invasive potential in gastric cancer. [J Natl Cancer Inst 85:402-407, 1993]