Theoretical considerations in using animal models of metastasis and brief methodology for in vivo colorectal cancer models in SCID and nude mice.

Metastatic spread is generally responsible for the mortality of colorectal cancer patients. There are no adequate treatments for advanced colorectal cancer, and novel therapeutic modalities are urgently required. To this end, valid metastatic models, which accurately mimic the disease process, are needed. When deciding upon a metastasis model, the goals of the investigation will dictate the complexity of the model chosen. If biological mechanisms are being investigated, only a small number of experimental animals may be required, and a more complex, surgically intensive model may be used. If a therapeutic agent is being evaluated, owing to group sizes required to generate statistically significant effects, a less complex, less surgically intensive model may be preferable. The latter, however, may encompass only a particular phase of metastasis rather than reflecting all aspects of the metastatic cascade.

Nuclear targeting of a midregion PTHrP fragment is necessary for stimulating growth in breast cancer cells.

Parathyroid-hormone related protein (PTHrP) is the primary factor in humoral hypercalcemia of malignancy and is highly secreted by breast cancers. The pro-hormone undergoes post-translational processing and cleavage to give rise to mature secretory peptides, one of which is midregion PTHrP (38-94/95/101) containing a nuclear localisation sequence (NLS) in amino acids (87-106). The current study investigates whether the NLS in midregion PTHrP is important in breast cancer growth. PTHrP-(67-101), a midregion PTHrP fragment containing NLS-(87-101) significantly increased growth of MCF-7 and MDA-MB231 cells (126.3 and 121.3% of control respectively in serum conditions), independent of PTHR1 whereas PTHrP-(67-86), which lacks the NLS did not. Fluorescent-labelled PTHrP-(67-101) translocated to the nucleus, whereas PTHrP-(67-86) remained cytosolic and a scrambled(+NLS) peptide was not internalised. In comparison, no growth influence or uptake was seen in non-tumour breast cells (Hs578Bst). Increases in intracellular calcium mobilisation were observed in breast cancer cells stimulated with both PTHrP-(67-101) and PTHrP-(67-86) (EC(50) of 3.2 pM and 2.2 pM respectively for MCF-7 cells), whereas inositide turnover was not detected. Both nuclear uptake and calcium signalling were attenuated in the presence of EGTA, but not with U73122 or N-terminal PTHrP peptides. Our studies indicate that the NLS-containing midregion PTHrP peptide is dependent on both internalisation and nuclear translocation to induce growth in breast cancer cells. These findings highlight the importance of midregion PTHrP and its receptor in breast cancer growth and may provide potential targets for future therapeutic intervention.

The biological and therapeutic importance of gastrin gene expression in pancreatic adenocarcinomas.

The gastrin gene is expressed widely in pancreatic adenocarcinomas and the study aimed to assess its role in both the resistance of cancer cells to apoptosis and the sensitivity of cells to chemotherapeutic agents. Two human pancreatic cell lines, PAN1 and BXPC3, expressed gastrin at both the RNA and protein levels and are shown to be representative of human pancreatic adenocarcinomas in terms of gastrin expression. Inhibition of endogenous gastrin production by tumor cells was achieved with neutralizing gastrin antiserum and transfection with a gastrin antisense plasmid. Gastrin antiserum synergized with both taxotere and gemcitabine in inhibiting the in vitro growth of the PAN1 cell line with the inhibitory effect of the antiserum increasing from 12.7% to 70.2% with taxotere (P < 0.05) and 28.6% with gemcitabine (P < 0.01) after controlling for the effects of the cytotoxics. Synergy was only achieved with taxotere in BXPC3 cells with the inhibitory effect of gastrin antiserum increasing from 22.9% to 50.0% (P < 0.005). Cells transfected with gastrin antisense had reduced in vitro growth in low serum conditions and were poorly tumorigenic in nude mice at an orthotopic site. Gastrin antisense-transfected PAN1 cells had increased sensitivity to the antiproliferative effects of both gemcitabine (IC50 of > 100 microg/ml reduced to 0.1 microg/ml) and taxotere (IC50 of 20 microg/ml reduced to < 0.01 microg/ml) when compared with vector controls. The increased sensitivity of PAN1 antisense coincided with increased caspase-3 activity and reduced protein kinase B/Akt phosphorylation in response to both gemcitabine and taxotere. Gastrin gene circumvention may be an optimal adjunct to chemotherapeutic agents, such as taxotere and gemcitabine, in pancreatic adenocarcinoma.

Expression and regulation of tissue inhibitor of metalloproteinase-1 and matrix metalloproteinases by intestinal myofibroblasts in inflammatory bowel disease.

Intestinal fibrosis and strictures frequently occur in Crohn's disease but not ulcerative colitis. We have recently shown that, compared to myofibroblasts obtained from normal and ulcerative colitis tissue, myofibroblasts isolated from fibrotic Crohn's disease mucosal samples express significantly lower amounts of transforming growth factor (TGF)-beta 3, but the expression of TGF-beta 2 was significantly greater. We now report that in myofibroblast cultures established from fibrotic Crohn's disease mucosal samples there is significantly higher constitutive expression of tissue inhibitor of metalloproteinase (TIMP)-1 compared to similar cells isolated from normal or ulcerative colitis tissue. Myofibroblasts derived from normal mucosa and from mucosa affected by ulcerative colitis or Crohn's disease also expressed matrix metalloproteinase (MMP)-1, MMP-2, and MMP-3 but did not express MMP-9. Recombinant (r) TGF-beta 1 and rTGF-beta 2, but not rTGF-beta 3, induced expression of TIMP-1 in normal intestinal myofibroblasts. These studies illustrate a potential mechanism by which differential expression of isoforms of TGF-beta may lead to excessive deposition of extracellular matrix and stricture formation via TIMP-1-mediated inhibition of MMP activity.