Protein kinase A regulates Lewis lung carcinoma adherence to extracellular matrix components and spontaneous metastasis.

Tumor cell adhesion to and migration through the extracellular matrix (ECM) can influence their capacity to disseminate. Since prior studies with Lewis lung carcinoma (LLC) tumors had shown metastatic clones to have more protein kinase A (PKA) activity than nonmetastatic clones, the present study assessed if PKA regulates the interaction between tumor and the ECM, and how this may be associated with the metastatic capacity of the tumor cells. This was accomplished with the use of metastatic (LLC-LN7) and nonmetastatic (LLC-C8) variants that had been stably transfected to overexpress the PKA Calpha subunit or to have blocked PKA activity. Cells with increased PKA activity were less adherent to vitronectin, laminin, and collagen I, and could more readily migrate through these ECM components than could transfectants with reduced PKA activity. PKA did not regulate adhesion to or migration through fibronectin, and did not appear to be associated with changes in expression of surface integrins. In addition to modulating tumor adhesion and migration in vitro, PKA activation caused an increased formation of metastases from s.c. tumors, but did not regulate formation of experimental metastases by i.v. injected tumor cells. These results suggest that PKA signaling is important for modulating the tumor-ECM interaction and can facilitate tumor transit from the primary tumor site.

Regulation of cytoskeletal organization in tumor cells by protein phosphatases-1 and -2A.

Non-metastatic Lewis lung carcinoma cells (LLC-C8) become more motile when protein phosphatases (PP-1 and -2A) are inhibited by okadaic acid, attaining the same level of motility as metastatic LLC (LLC-LN7) variants. This stimulation of LLC-C8 motility was tempered when protein kinase A activity was inhibited. We examined whether the okadaic acid-stimulated LLC-C8 motility was associated with alterations in the cytoskeletal organization so that these non-metastatic cells acquire the rounded morphology and diffuse cytoskeletal organization previously described for metastatic LLC-LN7 cells. Non-metastatic LLC-C8 are typically adherent during culture, achieving a spread morphology. Treatment of non-metastatic LLC-C8 cells with okadaic acid resulted in a contraction of most of their extended processes, formation of spikes and membrane blebs within 10 min, and complete cell rounding within 20 min for most of the cells. While the overall level of F-actin was minimally affected by the okadaic acid, its uniform distribution shifted to localization toward the periphery of the rounded cells, often concentrating at a single focus. Immunofluorescent staining for vimentin showed a similar shift to the cell periphery and similar capping. After okadaic acid treatment, the filamentous network of microtubules in non-metastatic LLC-C8 cells disappeared and was replaced with a diffusely staining distribution of beta-tubulin. These results show that PP-1 and -2A maintain cytoskeletal organization and that inhibition of this control reduces cytoskeletal organization and increases tumor cell motility.

Protein phosphatases limit tumor motility.

Elevators of cAMP, such as prostaglandin E2 (PGE2), activate protein kinase A (PKA) and induce PKA-stimulated motility and metastasis by metastatic Lewis lung carcinoma cells (LLC-LN7). Non-metastatic LLC (LLC-C8) are unresponsive to cAMP elevation even though they are not deficient in the PKA enzymes. To determine whether this PKA unresponsiveness might be due to increased dephosphorylation by serine/threonine protein phosphatases (PP-1/2A) within non-metastatic LLC-C8, the effects of the PP-1/2A inhibitor okadaic acid on the migration and invasion by non-metastatic LLC-C8 cells was measured. Okadaic acid stimulated motility of non-metastatic LLC-C8 cells to a level that was comparable to that of metastatic LLC-LN7 cells. PGE2 further increased the motility of the non-metastatic LLC-C8 cells when okadaic acid was present, although not in the absence of okadaic acid. The stimulation of motility by okadaic acid was diminished when PKA activity was inhibited. Dose-response studies with concentrations of okadaic acid that selectively inhibited PP-2A or both PP-2A and PP-1 showed a progressive increase in migration of non-metastatic LLC-C8 cells, suggesting that both PP-1 and PP-2A limit their motility. By contrast, metastatic LLC-LN7 cells were more motile than were non-metastatic LLC-C8 cells, but this motility was only marginally affected by okadaic acid. Comparisons of the levels of PP-1/2A enzyme activities in the LLC variants showed more activity in non-metastatic LLC-C8 than in metastatic LLC-LN7 cells. The identity of the PP whose activity was increased in the non-metastatic LLC-C8 was assessed by using okadaic acid, which selectively inhibits PP-2A activity at low concentrations and PP-1 and PP-2A at high concentrations, and calyculin A, which inhibits PP-2A at a similar concentration to that affected by okadaic acid but is more potent at inhibiting PP-1. The inhibition of PP activities by okadaic acid and by calyculin A showed a pattern which suggested the presence both of PP-1 and of PP-2A in non-metastatic LLC-C8 cells, but the presence of PP-1 and a reduction in PP-2A in metastatic LLC-LN7 cells. The sum of these data suggests that PKA-stimulated motility is restricted both by PP-1 and by PP-2A in non-metastatic LLC, and that a deficiency in this restriction results in increased migration and invasion.

Characterization of the primary gene product of rat incisor alpha-phosphophoryn.

The nature of the primary gene product for the alpha-phosphophoryn component of rat incisor dentin has been examined by cell-free translation of the total RNA and poly(A+) mRNA from rat maxillary incisors, including pulp cells and odontoblasts. The RNA was extracted by the guanidinium thiocyanate method and translated in a rabbit reticulocyte system. The translated proteins were analyzed by gradient gel electrophoresis, and alpha-phosphophoryn was identified by isolation on an anti-rat alpha-phosphophoryn antibody coupled Sepharose column and dot-blot procedures. The major protein identified as alpha-phosphophoryn had a molecular weight of 153 000 (+/- 5000) and had chromatographic properties similar to those of alpha-phosphophoryn. Since tissue-isolated rat phosphophoryn has a molecular weight of only approximately 90 000 when fully phosphorylated, it appears that the primary gene product is a prepro-alpha-phosphophoryn. Thus, alpha-phosphophoryn in the extracellular space of rat incisor dentin must be the product of one or more posttranslational proteolytic processing steps.

The dynamics of formation of a collagen-phosphophoryn conjugate in relation to the passage of the mineralization front in rat incisor dentin.

Dentin and predentin matrices contain Type I collagen and phosphophoryns as major constituents. A collagen-phosphophoryn conjugate is also present in small amounts. This conjugate has been implicated in the deposition of mineral. Its formation has been followed in rat incisors. Rats were labeled for varied time intervals with [3H]proline, followed by a 2-h pulse of [3H] serine. The soluble alpha- and beta-phosphophoryns were extracted under conditions minimizing degradation. The tooth residue was CNBr-treated and the collagen CNBr peptides alpha 1(I)CB7 and alpha 1(I)CB8 were collected along with the solubilized conjugate fraction. Each component was purified and the specific activities in [3H] proline, [3H]hydroxyproline, [3H]serine, and [3H]phosphoserine were determined. The collagen and alpha-phosphophoryn accumulated proline label linearly at the same rate over the entire period of labeling. Entry of [3H]proline into the conjugate fraction was delayed by approximately 9-10 h and then the label accumulated also linearly at the same rate. [3H]Serine was present at a different but constant level in each fraction; the conjugate had the lowest activity. These data indicate an extracellular formation of the conjugate at the mineralization front from precursors which followed different secretory pathways.