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Squamous cell carcinoma (SCC) antigen, including intracellular serine protease inhibitors, is widely used as a laboratory marker for cancers of squamous cell origin. Clinical evidences suggest that increased tissue-expression of SCC antigen predicts an invasive phenotype of cancer cells. Herein, we demonstrated that over-expression of SCC antigen increased the rate of EGF-stimulated cell migration. In the search for the underlying molecular mechanism, we have discovered that SCC antigen was translocated to the plasma membrane upon EGF stimulation and co-localized with polymerized-actin at lamellipodia. We further showed that, co-expression of Cdc42, a downstream target of the EGF receptor, enhanced translocation of the SCC antigen, while co-expression of dominant-inhibitory Cdc42 diminished its translocation. These results suggest that EGF-Cdc42 signal regulates the translocation of SCC antigen to the plasma membrane. Lamellipodia at the leading edge might be a site of action of SCC antigen.

Epidermal growth factor (EGF), phorbol esters (PEs), and retinoic acid (RA) inhibit differentiated functions of thyrocytes. In the present study the inhibitory effects of these growth-promoting factors on hormone synthesis were studied in thyroid follicles cultured in type-I collagen gel, and morphologic alteration by these factors was examined by light and electron microscopy (EM). Porcine open thyroid follicles obtained by treatment with 0.1% collagenase were embedded in collagen gel and cultured in Ham's F12 medium supplemented with 6H (insulin, hydrocortisone, somatostatin, transferrin, glycyl-his-lys, and thyrotropin) + 0.5% fetal bovine serum (FBS). After 1 week these open follicles developed to closed follicles, and the medium was changed to one containing 6H + 0.5% FBS + 0.1 microM sodium iodide (NaI). Some media were supplemented with either EGF, phorbol 12-myristate 13-acetate (PMA), or all-trans RA. The closed follicles retained ability for hormone synthesis for 2 weeks after the medium change in the presence of 6H + FBS + NaI. The amounts of T4 and T3 secreted into the culture medium from day 9 to day 12 after the medium change were 60% and 45% of those from day 0 to day 4, respectively. EGF reduced production of T4 and T3 by 61% and 69%, respectively; PMA, by 87% and 99%; and RA, by 55% and 44%. In the medium supplemented with 6H + 0.5% FBS, the follicles exhibited intact polarity. Apical surfaces with microvilli were oriented to the follicular lumen and tight junctions were on the apical side of cell-to-cell contacts. Desmosomes were found on both the apical and basal halves of the cell contacts.(ABSTRACT TRUNCATED AT 250 WORDS)

Porcine thyroid follicles cultured in suspension for 96 h synthesized and secreted thyroid hormones in the presence of thyrotropin (TSH). The secretion of newly synthesized hormones was assessed by determining the contents of thyroxine (T4) and triiodothyronine (T3) in the media and by paperchromatographic analysis of 125I-labelled hormones in the media where the follicles were cultured in the presence and absence of inhibitors of hormone synthesis. The hormone synthesis and secretion was modified by exogenously added NaI (0.1-100 microM). The maximal response was obtained at 1 microM. Thyroid peroxidase (TPO) activity in the cultured follicles with TSH for 96 h was dose-dependently inhibited by NaI. One hundred microM of NaI completely inhibited TSH-induced TPO activity. Moreover, both epidermal growth factor (EGF: 10(-9) and 10(-8) M) and phorbol 12-myristate 13-acetate (PMA: 10(-8) and 10(-7) M) inhibited de novo hormone synthesis. An induction of TPO activity by TSH was also inhibited by either agent. These data provide direct evidences that thyroid hormone synthesis is regulated by NaI as well as TSH at least in part via regulation of TPO activity and also that both EGF and PMA are inhibitory on thyroid hormone formation.

In an attempt to elucidate possible mechanism(s) for stimulated arachidonic acid metabolism by phorbol 12-myristate 13-acetate (PMA) and epidermal growth factor (EGF) in porcine thyroid cells, we examined the effects of protein kinase inhibitors, isoquinolinesulfonamide derivatives (H-7 and HA-1004), and cycloheximide. The production of PGE2 stimulated by either PMA or EGF was strongly inhibited by H-7, with an ID50 value of approximately 20 to 25 mumol/L in each case, as well as by cycloheximide, with an ID50 value of less than 0.5 micrograms/mL in each case. In contrast, 100 mumol/L of HA-1004 showed less inhibition of PGE2 production provocated by either PMA or EGF. On the other hand, PGE2 production in basal or stimulated condition by exogenously added arachidonic acid, was inhibited to an even lesser extent by both H-7 and cycloheximide. The EGF- and PMA-stimulated release of 3H-arachidonic acid from the cells was also strongly inhibited by H-7 and cycloheximide. These results suggest an induction of synthesis of some proteins responsible for the release of arachidonic acid, which might be attributed to protein kinase-C activation in arachidonic acid metabolism stimulated by PMA or EGF. Moreover, PGE2 production was potently induced by PMA and slightly by EGF in the cyclooxygenase-inactivated cells by acetyl salicylate pretreatment, which also suggests that both agents might induce the synthesis of cyclooxygenase in cultured porcine thyroid cells, although we did not measure its activity.

Effects of phorbol esters and epidermal growth factor (EGF) on prostaglandin E2 production by cultured porcine thyroid cells were examined. Both phorbol 12-myristate 13-acetate (PMA) and EGF stimulated prostaglandin E2 production by the cells in dose related fashion. PMA stimulated prostaglandin E2 production over fifty-fold with the dose of 10(-7) M compared with control. EGF (10(-7) M) also stimulated it about ten-fold. The ED50 values of PMA and EGF were respectively around 1 X 10(-9) M and 5 X 10(-10) M. Thyroid stimulating hormone (TSH), however, did not stimulate prostaglandin E2 production from 1 to 24-h incubation. The release of radioactivity from [3H]-arachidonic acid prelabeled cells was also stimulated by PMA and EGF, but not by TSH. These results indicate that both PMA and EGF are potent stimulators of prostaglandin E2 production, associated with the activity to stimulate arachidonic acid release in porcine thyroid cells.

The activity of thyroid peroxidase (TPO) in porcine follicles cultured for 96 h in suspension with five hormones (5H) still attained over 50% of that in the freshly isolated follicles. On the other hand, the activity in those cultured with 5H + TSH (6H) was several times higher than that cultured with 5H after 96 h, although an initial decrease of TPO activity during the first 24 h of culture was observed in both conditions. The ability of follicles to metabolize iodide (uptake and organification) when cultured with 6H for 96 h was also several times higher than that of those cultured with 5H. The half-maximal dose of TSH for stimulation of TPO activity and iodide metabolism was 0.03-0.04 mU/ml and the effect was mediated by cAMP. These results indicate that in porcine thyroid follicles in primary suspension culture, TPO activity as well as the ability of iodide metabolism is induced by chronic TSH stimulation. In addition, epidermal growth factor (EGF, 10(-9)M) and phorbol 12-myristate 13-acetate (PMA, 10(-8) M) completely inhibited TSH stimulation on both activities and also basal (5H) activity of iodide metabolism.