Beta-galactosidase staining on bone marrow. The osteoclast pitfall.

The enzyme beta-galactosidase, encoded by the bacterial gene lac-Z, is commonly used as a histochemical reporter to track transplanted cells in vivo or to analyze temporospatial gene expression patterns by coupling expression of specific target genes to beta-galactosidase activity. Previously, endogenous beta-galactosidase activity has been recognized as a confounding factor in the study of different soft tissues, but there is no description of the typical background on bone marrow sections when using the chromogenic substrate 5-Bromo-4-chloro-3-indolyl beta-D-Galactoside (X-Gal). In this report, we show that osteoclasts in bone marrow sections specifically and robustly stain blue with X-Gal. This leads to a typical background when bone marrow is examined that is present from the first day post partum throughout the adult life of experimental mice and can be confused with transgenic, bacterial beta-galactosidase expressing hematopoietic or stromal cells. Experimental variations in the X-Gal staining procedure, such as pH and time of exposure to substrate, were not sufficient to avoid this background. Therefore, these data demonstrate the need for strenuous controls when evaluating beta-galactosidase positive bone marrow cells. Verifiable bacterial beta-galactosidase positive bone marrow cells should be further identified using immunohistological or other approaches. Specifically, beta-galactosidase positive hematopoietic or stromal cells should be proven specifically not to be osteoclasts by co-staining or staining adjacent sections for specific markers of hematopoietic and stromal cells.

Epidermal growth factor receptor blockade by antibody IMC-C225 inhibits growth of a human pancreatic carcinoma xenograft in nude mice.

BACKGROUND: Pancreatic carcinoma is associated with a poor prognosis, and treatment options for patients with this disease are limited. The epidermal growth factor (EGF) receptor and its ligands are overexpressed in human pancreatic carcinoma and may contribute to the pathophysiology of these tumors. METHODS: The anti-EGF receptor monoclonal antibody IMC-C225 was used to determine the effects of EGF receptor blockade on the growth of human pancreatic carcinoma BxPC-3 cells in vitro. Athymic mice bearing established (200 mm(3)) subcutaneous BxPC-3 xenografts were treated with IMC-C225 (17 or 33 mg/kg every 3 days) alone or in combination with 5-fluorouracil (17 mg/kg twice weekly). RESULTS: IMC-C225 inhibited exogenous ligand-stimulated tyrosine phosphorylation of the EGF receptor on BxPC-3 tumor cells. Treatment of BxPC-3 cells with IMC-C225 inhibited DNA synthesis (23.8%) and colony formation in soft agar (45.6%). IMC-C225 treatment significantly suppressed the growth of BxPC-3 tumors compared with treatment with vehicle alone (P = 0.003). Combination therapy with IMC-C225 and the chemotherapeutic agent 5-fluorouracil enhanced the antitumor effects compared with either agent alone and resulted in regression of pancreatic tumors in several animals. Histologic examination of pancreatic tumors from mice treated with IMC-C225 showed extensive tumor necrosis that coincided with a substantial decrease in tumor cell proliferation and an increase in tumor cell apoptosis. CONCLUSIONS: These data suggest that IMC-C225 affects the growth of pancreatic tumors by inhibiting EGF receptor-dependent proliferation and survival, and demonstrates the potential for therapeutic application of IMC-C225 antibody in the treatment of human pancreatic carcinoma.