Selective distribution of protein kinase A regulatory subunit RII{alpha} in rodent gliomas

Abstract

Differential diagnosis of brain tumor types is mainly based on cell morphology and could benefit from additional markers. The cAMP second-messenger system is involved in regulating cell proliferation and differentiation and is conceivably modulated during cancer transformation. The cAMP second-messenger system mainly activates protein kinases, which are in part docked to cytoskeleton, membranes, or organelles by anchoring proteins, forming protein aggregates that are detergent insoluble and not freely diffusible and that are characteristic for each cell type. The intracellular distribution of the detergent-insoluble regulatory subunits (R) of the cAMP-dependent protein kinase has been examined in mouse and rat glioma cells both in vitro and in vivo by immunohistochemistry. In normal rodent brains, the RIIalpha regulatory subunit is detergent insoluble only in ependymal cells, while in the rest of the brain it is present in soluble form. Immunohistochemistry shows that in both mouse and rat glioma cell lines, RIIalpha is mainly detergent insoluble. RIIalpha is localized close to the nucleus, associated with smooth vesicles in the trans-Golgi network area. Both paclitaxel and vinblastine cause a redistribution of RIIalpha within the cell. Under conditions that increased intracellular cAMP, apoptosis of glioma cells was observed, and it was accompanied by RIIalpha redistribution. Also in vivo, detergent-insoluble RIIalpha can be observed in mouse and rat gliomas, where it delineates the border between normal brain tissue and glioma. Therefore, intracellular distribution of detergent-insoluble RIIalpha can assist in detecting tumor cells within the brain, thus making the histologic diagnosis of brain tumors more accurate, and may represent an additional target for therapy.

Fig. 1.

RIIα distribution in mouse and rat glioma. (A) Western blot of mouse primary glial cell and GL261 cells. Lanes 1–4 are probed against RI, lanes 5–8 against RII. Both proteins are present in Triton-insoluble pellets. M, molecular weight markers: 1 and 5, soluble fraction from glial cell primary culture; 2 and 6, insoluble fraction from glial cell primary culture; 3 and 7, GL261 soluble fraction; 4 and 8, GL261 insoluble fraction. (B) Percentage of apoptosis (+ SEM) in GL261 cells treated with various agents. All treated cells present a statistically significant increase in apoptosis (analysis of variance, p < 0.05), compared to control cells. (C) In a GL261 cell, RIIα immunostaining (green) appears as a cluster of small dots on one side of the cell nucleus (blue). (D) RIIα (green) localization in GL261 cells is not coincident with the intermediate filament vimentin (red). Scale bar, 10 μm for C and D. IBMX, isobuytlmethylxantine; 8Br-cAMP, 8 bromo-cAMP; DB-cAMP, 6-dibutyryl-cAMP.

Fig. 2.

RIIα and golgin distribution in rat glioma cells. (A) Detail of an F98 cell doubly immunolabeled for RIIα and golgin: differential interference contrast image of cells (far left), RIIα (green) and golgin (red) staining, and the superimposed image (far right). The two signals (yellow) are present in the same subcellular compartment. Scale bar, 10 μm. (B) Transmission electron micrograph of a GL261 cell, immunolabeled with anti-RIIα antibody. The gold grains are mostly distributed at the periphery of Golgi vesicles (v), whereas the nucleus (N) and mitochondria (m) are devoid of signal. Scale bar, 0.5 μm. (C) Transmission electron micrograph of a smooth vesicle in a GL261 cell, doubly immunolabeled for RII (larger gold grains, black arrow) and golgin (smaller grains, open arrow). The vesicle appears to be labeled by both antibodies.

Fig. 3.

RIIα distribution in GL261 cells. (A) In untreated cells, RIIα (red) appears localized on one side of the cell and does not overlap with the astroglia-specific intermediate filament glial fibrillary acidic protein (GFAP; green). (B) GL261 cells, after treatment with the diterpene activator of adenylate cyclase, forskolin, which stimulates cAMP production. RIIα (red) is apparently more loosely distributed around the nuclei. GFAP (green) strongly stains cellular processes. (C) GL261 cells treated with H89, an inhibitor of cAMP-dependent protein kinase catalytic activity. RIIα (red) appears scattered around the nuclei, in particular in cells which lack GFAP expression. If present, the GFAP (green) filaments are not clearly defined. (D) GL261 cells after paclitaxel treatment. RIIα (red) is hardly detectable around cell nuclei, and vimentin (green) lacks the filamentous appearance in the few cells that are surviving. Scale bar, 10 μm for all panels.

Fig. 4.

RIIα distribution in GL261 cells and mouse glioma. (A) GL261 cells treated with vinblastine. RIIα (red) appears scattered all around the nucleus. (B) GL261 cells treated with vinblastine. RIIα (red) appears scattered all around the nucleus, whereas vimentin staining (green) appears not affected by the treatment. (C) Hematoxylin-eosin staining of a mouse brain hosting a glioma (left). The border of the tumor (arrow) can be easily identified. (D) RIIα (red) also highlights the tumor border (arrow). The transition from tumor to normal parenchyma is easier to visualize than in hematoxylin and eosin-stained sections (C). The cells inside the tumor mass are labeled, as well as some isolated RIIα-labeled cells that appear at the border, surrounded by unlabeled tissue. Their detection is easier than in hematoxylin and eosin-stained sections (see C). Scale bar, 10 μm for A and B; 50 μm for C and D.

Fig. 5.

RIIα distribution in mouse and rat glioma. (A) Glioma obtained from a mouse after GL261 inoculation in vivo. RIIα (green) is distributed in clusters of small dots localized near nuclei (blue). Their appearance resembles those detected in cultured glioma cells. (B) Distribution of RIIα (red) in F98 cells resembles those seen in GL261 cells, consisting of a cluster of dots located on one side of each nucleus (blue). (C) Glioma obtained from a rat after F98 inoculation in vivo. RIIα (red) is distributed in clusters of small dots localized near nuclei (blue). (D) Glioma obtained from a rat after inoculation of F98 cells. As in the mouse, RIIα (red) highlights the tumor border (arrow). (E) RIIα immunolabeling (red) of the human glioblastoma cells Gli36: the labeling is similar to that in the mouse and rat glioma cell lines. The nucleus is stained blue. Scale bar, 10 μm for A, B, C, and E; 25 μm for D.