We determined whether the implantation of human pancreatic cancer cells into the pancreas of nude mice can be used to select variants with increasing metastatic potential. COLO 357 line fast-growing cells were injected into the spleen or pancreas of nude mice. Hepatic metastases were harvested, and tumor cells were reinjected into the spleen or pancreas. This cycle was repeated several times to yield cell lines L3.6sl (spleen to liver) and L3.6pl (pancreas to liver). The variant cells produced significantly higher incidence and number of lymph node and liver metastases than the parental cells. Their increased metastatic potential was associated with increased expression (mRNA and protein) of the proangiogenic molecules basic fibroblast growth factor, vascular endothelial growth factor, and interleukin-8. The metastatic cells also exhibited increased motility and invasiveness, which were associated with increased expression of collagenase type IV (MMP-9) and decreased expression of E-cadherin. Collectively, the data show that the orthotopic implantation of human pancreatic cancer cells in nude mice is a relevant model with which to study the biology of pancreatic cancer metastasis and to select variant cell lines with enhanced metastatic potential.

cAMP response element-binding protein (CREB) and activating transcription factor 1 (ATF-1), members of the CREB/ATF family, have been implicated in cAMPand calcium-induced transcriptional activation. We have previously demonstrated that quenching of CREBassociated proteins in metastatic melanoma cells by a dominant-negative CREB (KCREB) that is mutated within its DNA-binding domain decreased their radiation resistance, and their tumorigenic and metastatic potential in nude mice. As the induction of apoptosis by diverse exogenous signals is dependent on the elevation of intracellular Ca 2؉ , the purpose of this study was to determine the role of CREB and its associated proteins in apoptosis using KCREB. We used thapsigargin (Tg), which inhibits endoplasmic reticulum-dependent Ca 2؉ -ATPase and thereby increases cytosolic Ca 2؉ , to induce apoptosis. MeWo human melanoma cells were transfected with the KCREB expression vector and subsequently analyzed for their susceptibility to Tg-induced apoptosis. Here we demonstrate that expression of KCREB in MeWo cells rendered them susceptible to Tginduced apoptosis. Tg treatment induced phosphorylation of CREB and possibly ATF-1 transcription factors. Treatment with Tg induced CRE-dependent transcription in parental cells, whereas this activation was reduced in the KCREB-transfected cells. In addition, CAT activity driven by the CRE-dependent promoter was inhibited in parental MeWo cells cotransfected with increasing concentrations of KCREB in a dose-dependent manner. We did not observe any changes in Bcl-2 or Bcl-2-related proteins (Bcl-x, Bax, and Bad) in control or KCREB-transfected cells before or after treatment with Tg. Collectively, these data indicate that CREB and its associated proteins act as survival factors for human melanoma cells, and hence contribute to the acquisition of the malignant phenotype.The molecular basis of human malignant melanoma progression has remained largely unknown, despite the fact that the worldwide incidence of melanoma is increasing more than that of any other neoplastic disease (1). The development of malignant melanoma in humans progresses through a multistage process. The switches from melanocyte to nevi, to radial growth, and subsequently to vertical growth phase (metastatic phenotype) are associated with decreased dependence on growth factors, diminished anchorage dependence, and reduced contact inhibition (2, 3).A large body of data concerning the molecular control of melanoma progression has come from studies using mitogens. In culture, melanocytes synergistically respond to a number of growth factors, which in combination with each other or with 12-O-tetradecanoylphorbol-13-acetate or cAMP stimulate not only proliferation but also pigmentation (4). These growth factors include several fibroblast growth factors, hepatocyte growth factor, and stem cell factor (also known as KIT ligand, MGF, and steel factor), all of which stimulate receptors tyrosine kinase. As melanocyte proliferation and differentiation are positively regulated ...

Vascular endothelial growth factor (VEGF) is the major pro-angiogenic factor for most tumors. VEGF expression has been shown to be associated with a poor prognosis in human pancreatic cancer. The purpose of our study was to determine the effect of blockade of VEGF receptor-2 activity with or without gemcitabine on tumor growth and metastasis in an orthotopic model of human pancreatic cancer in nude mice. Therapy with gemcitabine or DC101, a VEGF receptor-2 antibody, resulted in a significant reduction of primary pancreatic tumor growth compared to untreated controls. The combination of DC101 and gemcitabine inhibited primary pancreatic tumor growth and lymphatic metastasis to a greater degree than either agent alone. Treatment with DC101 decreased vessel counts and increased the area of hypoxic tumor tissue compared to controls. Immunofluorescent double staining for apoptotic endothelial cells demonstrated a significant increase in the number apoptotic endothelial cells 24 days after initiation of therapy with DC101 plus gemcitabine. DC101 plus gemcitabine also increased tumor cell death and decreased tumor cell proliferation in pancreatic tumors. These findings indicate that blockade of VEGF receptor activation interferes with the survival of tumor endothelial cells, resulting in a reduction of primary pancreatic tumor growth in nude mice. Furthermore, the data demonstrate that anti-VEGF receptor-2 therapy potentiates the tumoricidal effect of gemcitabine in this model. Anti-VEGF receptor-2 therapy in combination with gemcitabine may be a novel therapeutic approach for advanced pancreatic cancer.

Tumor necrosis factor (TNF) exerts cytotoxicity on many types of tumor cells but not on normal cells. The molecular events leading to cell death triggered by TNF are still poorly understood. Our previous studies have shown that enforced expression of an activated H‐ras oncogene converted non‐tumorigenic, TNF‐resistant C3H 10T1/2 fibroblasts into tumorigenic cells that also became very sensitive to TNF‐induced apoptosis. This finding suggested that Ras activation may play a role in TNF‐induced apoptosis. In this study we investigated whether Ras activation is an obligatory step in TNF‐induced apoptosis. Introduction of two different molecular antagonists of Ras, the rap1A tumor suppressor gene or the dominant‐negative rasN17 gene, into H‐ras‐transformed 10TEJ cells inhibited TNF‐induced apoptosis. Similar results were obtained with L929 cells, a fibroblast cell line sensitive to TNF‐induced apoptosis, which does not have a ras mutation. While Ras is constitutively activated in TNF‐sensitive 10TEJ cells, TNF treatment increased Ras‐bound GTP in TNF‐sensitive L929 cells but not in TNF‐resistant 10T1/2 cells. Moreover, RasN17 expression blocked TNF‐induced Ras‐GTP formation in L929 cells. These results demonstrate that Ras activation is required for TNF‐induced apoptosis in mouse fibroblasts.

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