Calcium signals in prostate cancer cells: specific activation by bone-matrix proteins.

Cancer of the prostate commonly metastasizes to bony sites where cells acquire an aggressive, rapidly proliferating, androgen-independent phenotype. The interaction between bone and prostate, thus, becomes a key factor in disease progression. Fluctuations in intracellular ionized Ca2+ [Ca2+]i are rapid, regulated signal transduction events often associated with cell proliferation. Hence, Ca2+ signals provide a convenient measure of early events in cancer cell growth. This study developed single cell fluorescent imaging techniques to visualize Ca2+ signals in Fura-2 loaded prostatic cancer cell lines of various metastatic phenotypes. Solubilized bone fractions containing extracellular matrix and associated proteins were tested for the ability to trigger Ca2+ signals in prostate cancer cell lines. Fractions representing the complete repertoire of non-collagenous proteins present in mineralized bone were tested. Results demonstrated that two bone fractions termed D3b- and D4a-triggered Ca2+ signals in prostate cancer cells derived from bone (PC-3), but not brain (DU-145) metastases of prostate cancer. Lymph-node derived LNCaP cells also did not produce a Ca2+ signal in response to addition of soluble bone matrix. No other bone fractions produced a Ca2+ signal in PC-3 cells. It is of interest that bone fractions D3b and D4a contain a number of non-collagenous matrix proteins including osteonectin (SPARC) and osteopontin (OPN), as well as prothrombin. Moreover, antibody LM609 that recognizes the alpha v beta 3 integrin, blocks the ability of OPN to trigger a Ca2+ transient in PC-3 cells. These studies support a conclusion that bone-matrix proteins play a role in the growth and progression of metastatic prostate cancer, and that prior growth in bone may be associated with development of a bone-matrix-responsive phenotype.

Osteopontin: possible role in prostate cancer progression.

Human prostate cancer has the propensity to metastasize to the bone where reciprocal cellular interactions between prostate cancer and bone cells are known to occur. Osteopontin (OPN), a noncollagenous bone extracellular matrix, is a secreted adhesive glycoprotein with a functional RGD cell-binding domain that interacts with the alpha(v)beta3 cell surface integrin heterodimer. OPN has been associated with malignant transformation as well as being ligand to the CD44 receptor. Polyclonal antibodies to human OPN (hOPN) were prepared, and specificity was shown by preabsorption with recombinant hOPN. The stimulatory effect of hOPN protein and the inhibitory effect of hOPN antibody on human prostate cancer cell lines LNCaP and C4-2 were assessed by induction or inhibition of anchorage-independent growth, respectively. Expression of hOPN mRNA in prostate cancer cell lines and human prostate cancer tissue specimens were measured by mRNA blot analysis. Protein expression was assessed by immunohistochemistry in human prostate cancer specimens and by Western blot analysis in prostate cancer cell lines. hOPN stimulated anchorage-independent growth of the human prostate cancer cell lines LNCaP and C4-2 in vitro. Antibodies to hOPN inhibited the growth-stimulatory effect by endogenous OPN, which can be overcome by the addition of exogenous hOPN. hOPN mRNA and protein are expressed in human prostate cancer cell lines in vitro and in clinical human prostate cancer specimens. These findings taken together suggest that OPN may act as a paracrine and autocrine mediator of prostate cancer growth and progression.

Osteopontin (OPN) distribution in premalignant and malignant lesions of oral epithelium and expression in cell lines derived from squamous cell carcinoma of the oral cavity.

The objectives of this study were to assess the immunolocalization of human osteopontin (OPN) in oral lesions and to identify human cell lines of oral squamous cell carcinoma (OSCC) origin that express OPN mRNA. OPN was localized using immunohistochemistry in the following oral specimens: normal epithelium (n=6), epithelial hyperplasia (n=4), epithelial dysplasia (n=28), carcinoma in situ (n=11) and squamous cell carcinoma (n=43). Cell lines UMSCC-1, MDA TU 138, MDA 686LN, SCC4, SCC9, SCC25, CAL 27 and MDA 1483 were characterized for OPN mRNA expression using Northern blotting. OPN was not detected in normal oral epithelium. Intracellular and intercellular immunoreactivity was seen in 75% of hyperplasias, 57% of dysplasias, 54% of carcinoma in situ and 67% of squamous cell carcinomas. UMSCC-1 expressed high levels of OPN mRNA. We conclude that OPN protein is detectable in premalignant and malignant lesions arising from oral epithelium. UMSCC-1 may be a useful cell line in which to conduct in vitro studies designed to clarify the role of OPN in OSCC.

Improved immunohistochemical staining of osteopontin (OPN) in paraffin-embedded archival bone specimens following antigen retrieval: anti-human OPN antibody recognizes multiple molecular forms.

Studies to assess osteopontin (OPN) localization in adult human bone using immunochemical techniques produce conflicting results due to variations in tissue processing or antibody immunoreactivity. The present study was designed to resolve these discrepancies using well-characterized antibodies and improved antigen detection. An anti-osteopontin (alpha-OPN) antiserum was developed that recognizes various soluble molecular weight forms of human OPN, including monomeric, cleaved, and dimerized products. An affinity column of full length recombinant human OPN (rOPN) coupled to support was used to purify alpha-OPN antibodies. Western analysis showed that the affinity-purified antibodies recognized numerous molecular weight forms of OPN. These antibodies were used to study the distribution of OPN in adult human bone using immunohistochemical techniques combined with an antigen retrieval protocol utilizing a newly developed antigen retrieval solution, Retriev-Alltrade mark (Bronco Technologies Inc, Pasadena, TX). Immunolocalization of OPN in archival bone specimens prior to antigen retrieval produced no demonstrable immunostaining even at high concentrations of alpha-OPN. Use of the antigen retrieval protocol restored OPN immunoreactivity, with strong staining apparent in cement lines, osteoblasts, osteocytes, canaliculi, osteoid, and bone matrix. We conclude that antigen retrieval restores immunochemical recognition of OPN in archival specimens containing bone without increasing nonspecific binding.

Bone matrix proteins in osteogenesis and remodelling in the neonatal rat mandible as studied by immunolocalization of osteopontin, bone sialoprotein, alpha 2HS-glycoprotein and alkaline phosphatase.

The neonatal rat mandible was used as a model to study bone formation, mineralization, quiescence, and resorption, using immunolocalization and a variety of tissue-processing techniques. Monospecific antibodies for osteopontin (OPN), bone sialoprotein (BSP), alkaline phosphatase (AP) and alpha 2HS-glycoprotein (alpha 2HS-GP) were used on fixed paraffin-embedded tissue, fixed frozen tissue and unfixed frozen tissue. Immunostaining was correlated with mineral content by two procedures, the von Kossa and the morin techniques. Morin fluorescence was used with secondary immunostaining to provide a way of closely correlating bone matrix proteins and matrix mineralization. Co-immunolocalization procedures were used to compare the sites of bone proteins in the matrix. AP was found earliest during osteogenic cell differentiation, appearing in the preosteoblasts, followed by OPN and BSP, which first appeared in osteoblasts. alpha 2HS-GP expression was not observed in cells. The results provide clear evidence for the presence of OPN in osteoid, while BSP and alpha 2HS-GP were confined to the mineralized matrix. Immunostaining of bone proteins is highly technique-dependent: immunolocalization investigations required several methods of approach to ensure adequate demonstration of these proteins in cells and matrix. The results support the contention that osteopontin is multifunctional in bone metabolism, and that alpha 2HS-GP, though produced in the liver, is abundant in bone matrix and may also have a function in bone metabolism.