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The epidermal growth factor (EGF) receptor (EGFR) is activated by EGF and other EGF-like growth factors, including amphiregulin (AR). We characterized the localization and mitogenic action of AR in T3M4 and COLO-357 human pancreatic cancer cell lines and determined whether the presence of AR in human pancreatic cancers correlates with patient survival. Both T3M4 and COLO-357 cells were found to be extremely sensitive to AR, one-half maximal stimulation occurring at a concentration of 70 and 50 pM, respectively. The magnitude of the stimulatory effect was greater with AR than with EGF. Both cell lines exhibited AR immunostaining, which was present in a variable manner in the cytoplasm, nucleus and nucleoli. Immunohistochemical analysis of 62 pancreatic cancer tissues revealed the presence of nuclear and/or cytoplasmic AR immunoreactivity in the cancer cells. Cytoplasmic, but not nuclear localization of AR in the pancreatic cancer cells was associated with a statistically significant decrease in the post-operative survival period. The presence of EGFR alone in the cancer cells did not correlate with decreased survival, whereas coexpression of cytoplasmic AR and EGFR was associated with shorter survival. Distant metastases did not always exhibit cytoplasmic AR immunoreactivity. These findings point to the existence of an EGFR: AR autocrine loop in human pancreatic cancer which may contribute to its biological aggressiveness.

The murine double minute-2 (MDM2) gene encodes a 90 kDa protein which binds and inactivates the protein product of the p53 tumor suppressor gene. To elucidate the potential role of MDM2 in benign and malignant hyperproliferative conditions in the pancreas, we studied MDM2 expression in cultured human pancreatic cancer cells and in pancreatic tissues from normal donors, patients with pancreatic ductal adenocarcinoma and patients with chronic pancreatitis (CP). All the tested cell lines (PANC-1, COLO-357, HPAF and T3M4) expressed a 5.5 kilobase MDM2 mRNA transcript and exhibited nuclear MDM2 immunostaining. MDM2 mRNA levels were comparable in all 18 normal and 14 CP tissues for which RNA samples were available for analysis. By comparison with the normal samples, MDM2 mRNA levels were increased 6.4-fold in 8 of 12 human pancreatic cancer samples (p < 0.0001). All 8 samples exhibited nuclear MDM2 immunostaining, which was also present in 24 of 37 additional pancreatic cancers. Mild MDM2 immunoreactivity was seen in only 1 of 20 CP samples and in none of the 18 normal samples. These findings indicate that MDM2 is overexpressed in a majority of pancreatic adenocarcinomas, but not in CP samples. This selective overexpression raises the possibility that MDM2 may contribute to pancreatic neoplastic transformation by interfering with the growth-suppressing activity of wild-type p53.

Immunohistochemical analysis of the p53 tumor suppressor gene was performed in 69 human pancreatic ductal adenocarcinomas, using a highly specific anti-p53 antibody. Nuclear immunoreactivity was found in 40 tumors, yielding an overall frequency of 58%. Immunoblotting confirmed that nuclear immunoreactivity was associated with increased p53 protein levels. p53 mRNA levels were increased in 9 of 9 tested cancers, without evidence for gene amplification. Analysis of the immunostaining data by chi-square and log-rank indicated that the presence of nuclear immunoreactivity correlated with a more advanced clinical stage, and a statistically significant decrease in the post-operative survival period. In 12 cancers, metastatic tissue samples were also available for p53 analysis. Nuclear p53 immunostaining in the primary tumors was not always associated with p53 immunoreactivity in the metastatic samples, and metastases occurred in the absence of nuclear p53 immunoreactivity in the primary lesion. These findings suggest that increased p53 protein levels in human pancreatic cancer may be due not only to p53 mutations which attenuate the degradation of the protein but also to an increase in p53 mRNA levels leading to increased p53 synthesis, and that p53 nuclear immunoreactivity in these cancers implies enhanced tumor aggressiveness but is not essential for the development of metastases.

Heparin-binding EGF-like growth factor (HB-EGF) is a polypeptide with an apparent molecular weight of 22 kilodalton that is related to epidermal growth factor (EGF) and that binds and activates the EGF receptor. We examined HB-EGF biological action and expression in human pancreatic cancer cell lines, and compared HB-EGF expression in normal and cancerous pancreatic tissues. HB-EGF enhanced the growth of human pancreatic cancer cells in a dose-dependent manner. Several cell lines expressed HB-EGF mRNA transcripts, and the transcript level was enhanced by HB-EGF, as well as by 12-O-tetradecanoylphorbol-13-acetate and transforming growth factor-alpha (TGF-alpha). By comparison with the normal pancreas, HB-EGF mRNA levels were increased in human pancreatic cancer tissues. These findings suggest that HB-EGF may participate in aberrant autocrine and paracrine activation of the EGF receptor, thereby contributing to pancreatic cancer cell growth.

To determine the influence of the underlining disease on the duration of total parenteral nutrition (TPN) and on the long-term prognosis, data from 30 admitted patients with permanent (n = 23) and temporary (n = 7 short bowel syndrome were retrospectively analyzed and the present status of all living patients evaluated. Patients with "permanent short bowel" after thrombosis of the superior mesenteric artery (group I, n = 13) or malignant tumours (group II, n = 3) had a decreased survival compared to patients with other causes such as ileus, intestinal volvulus-thrombosis of mesenteric veins or benign tumors (group III, n = 7). Within the first six postoperative months, all patients in group I and all patients in group II died of the underlying disease whereas none died in group III. One patient in group I and one patient in group III died as a result of complications related to TPN. The different underlying diseases had no influence on the adaptation of the small intestine or on the duration of TPN in the surviving patients. Return to enteral autonomy seems to mainly depend on the length of the remaining small and large bowel and early enteral feeding. Complications of parenteral nutrition and possible options for the surgical treatment of the short bowel syndrome are discussed.

Acidic fibroblast growth factor (aFGF) and basic FGF (bFGF) are mitogenic polypeptides that may contribute to cancer cell proliferation. In the present study we examined aFGF and bFGF expression in human pancreatic cancer. Northern blot analysis of total RNA isolated from 12 pancreatic cancers revealed elevated aFGF and bFGF mRNA levels in 12 and 10 samples, respectively, by comparison with the normal human pancreas. Immunostaining demonstrated the presence of aFGF and bFGF in many cancer cells and in the atrophic acini and ducts adjacent to the cancer cells, but to a much lesser extent in the surrounding fibroblasts. By in situ hybridization, both mRNA moieties colocalized with their respective proteins and were abundant in many cancer cells. Immunoblotting confirmed that cancer tissues with increased aFGF and bFGF immunoreactivity contained elevated levels of both proteins. To determine the significance of aFGF and bFGF expression in the pancreatic cancer cells, immunohistochemical analysis of 78 human pancreatic carcinomas was performed. aFGF and bFGF immunoreactivity was present in the cancer cells in 47 (60%) and 44 (56%) of the tumors, respectively. There was a significant correlation between the presence of either aFGF or bFGF in the cancer cells and advanced tumor stage, and the presence of bFGF and shorter patient survival. These data suggest that aFGF and bFGF are overexpressed in a significant proportion of human pancreatic carcinoma cells and that this overexpression may contribute to disease progression.

Pancreatic expression of the p53 tumor suppressor gene was studied in pancreatic adenocarcinomas and chronic pancreatitis. By immunohistochemistry, 16 of 34 (47%) cancers and none of the 24 chronic pancreatitis samples revealed nuclear staining. Sequence analysis indicated that 8 of 24 (33%) cancers were mutated for the p53 gene. Point substitutions occurred at codons 35, 105, 133, 213, 213, 258, and 299. A three base-pair in-frame insertion was identified between codons 261 and 262. None of 8 chronic pancreatitis samples exhibited p53 gene mutations. These data support a role for p53 gene alterations in human pancreatic cancer, and suggest that loss of its regulatory functions may constitute one of the differences between pancreatic cancer and chronic pancreatitis.

Immunohistochemical analysis for the epidermal growth factor receptor (EGFR), EGF and transforming growth factor-alpha (TGF-alpha) was performed in 87 human pancreatic carcinomas. Expression frequencies for EGFR, EGF, and TGF-alpha were 43%, 46% and 54%, respectively. Coexpression of the receptor and at least one of its ligands occurred in 38% of the tumors, and correlated with large tumor size, advanced clinical staging, and decreased survival period. In situ hybridization revealed that the respective mRNAs were also overexpressed in the carcinomas. These findings suggest that coexpression of EGFR and its ligands may contribute to the aggressiveness of human pancreatic cancer.