P arathyroid hormone-related protein (PTHrP) was initially isolated from tumors associated with humoral hypercalcemia of malignancy and has since been identified in many normal tissues, in which it appears to act as an auto/paracrine growth/differentiation factor (Philbrick et al. 1996).Neurons from different subregions of the rat brain, including the cerebellum and the hippocampus, express PTHrP and PTH/PTHrP receptors (Weir et al. 1990;Ureña et al. 1993). In the rat, PTHrP has also been detected in the meninges but not in astrocytes that have PTH/PTHrP receptors (Struckhoff and Turzynski 1995). We evaluated the presence of PTHrP in human astrocytomas, using immunohistochemistry and Western immunoblot analysis.Four low-grade astrocytomas and five high-grade astrocytomas (Daumas-Duport et al. 1988) were studied. Fresh tumor samples were fixed overnight in 10% buffered formalin and embedded in paraffin before sectioning. The histological features were evaluated on hematoxylin-and eosin-stained tumor sections. Cytologically, the constituent tumor cells varied in size and in histological appearance. They consisted of small cells with hyperchromatic nuclei and larger cells with glassy cytoplasm and the morphological appearance of astrocytes.Immunostainings were performed with anti-PTHrP antisera C6 and C13, recognizing the C-and N-terminal region of PTHrP, respectively (Albar et al. 1996), and MIB 1 monoclonal specific antibody (Immunotech; Marseille, France), recognizing the Ki-67 proliferation marker, at 1:100 dilution, by the avidin-biotinperoxidase complex method. Paraffin-embedded tissue sections (5 m) were mounted on glass slides pretreated with 1 g/liter poly-l -lysine hydrobromide (Sigma; St Louis, MO). After heating at 60 Њ C for 12-24 hr, tissue samples were deparaffinized with xylene and rehydrated through a series of graded ethanols. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 5 min. Then the tissue sections for PTHrP staining were treated with 0.1% trypsin for 5 min and then blocked with 10% nonspecific porcine serum for 10 min. Tissue samples for MIB 1 staining, in 10 mM sodium citrate buffer, pH 6, were boiled for 2 min under pressure. Incubations were carried out in a humidified chamber for 2 hr at room temperature (RT). Positive staining was developed with 3,3 Ј -diaminobenzidine (Sigma) for 10 min. As negative controls, some tumor sections were incubated with saline buffer instead of the primary antiserum.Positive immunostaining for PTHrP was found in all nine astrocytomas with antisera C6 and C13. With either antiserum, the staining was diffuse in the cytoplasm of neoplastic cells and was prominent in the larger cells with a more astrocytic appearance ( Figures 1A and 1B). These cells were abundant in the five high-grade astrocytomas, whose proliferative activity, expressed as percentage of MIB 1-positive nuclear area was (mean Ϯ SD) 17 Ϯ 5%, greater than 5 Ϯ 3% in the four low-grade cases. Adjacent non-neoplastic cortex in the tissue samples did not stain for PTHrP. ...
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