Vascular tube formation on matrix metalloproteinase-1-damaged collagen.

Connective tissue damage and angiogenesis are both important features of tumour growth and invasion. Here, we show that endothelial cells maintained on a three-dimensional lattice of intact polymerised collagen formed a monolayer of cells with a cobblestone morphology. When the collagen was exposed to organ culture fluid from human basal cell tumours of the skin (containing a high level of active matrix metalloproteinase-1 (MMP-1)), degradation of the collagen matrix occurred. The major degradation products were the $3 over 4$- and $1 over 4$-sized fragments known to result from the action of MMP-1 on type I collagen. When endothelial cells were maintained on the partially degraded collagen, the cells organised into a network of vascular tubes. Pretreatment of the organ culture fluid with either tissue inhibitor of metalloproteinase-1 (TIMP-1) or neutralising antibody to MMP-1 prevented degradation of the collagen lattice and concomitantly inhibited endothelial cell organisation into the vascular network. Purified (activated) MMP-1 duplicated the effects of skin organ culture fluid, but other enzymes including MMP-9 (gelatinase B), elastase or trypsin failed to produce measurable fragments from intact collagen and also failed to promote vascular tube formation. Together, these studies suggest that damage to the collagenous matrix is itself an important inducer of new vessel formation.

Inhibition of type I procollagen synthesis by damaged collagen in photoaged skin and by collagenase-degraded collagen in vitro.

Type I and type III procollagen are reduced in photodamaged human skin. This reduction could result from increased degradation by metalloproteinases and/or from reduced procollagen synthesis. In the present study, we investigated type I procollagen production in photodamaged and sun-protected human skin. Skin samples from severely sun-damaged forearm skin and matched sun-protected hip skin from the same individuals were assessed for type I procollagen gene expression by in situ hybridization and for type I procollagen protein by immunostaining. Both mRNA and protein were reduced ( approximately 65 and 57%, respectively) in photodamaged forearm skin compared to sun-protected hip skin. We next investigated whether reduced type I procollagen production was because of inherently reduced capacity of skin fibroblasts in severely photodamaged forearm skin to synthesize procollagen, or whether contextual influences within photodamaged skin act to down-regulate type I procollagen synthesis. For these studies, fibroblasts from photodamaged skin and matched sun-protected skin were established in culture. Equivalent numbers of fibroblasts were isolated from the two skin sites. Fibroblasts from the two sites had similar growth capacities and produced virtually identical amounts of type I procollagen protein. These findings indicate that the lack of type I procollagen synthesis in sun-damaged skin is not because of irreversible damage to fibroblast collagen-synthetic capacity. It follows, therefore, that factors within the severely photodamaged skin may act in some manner to inhibit procollagen production by cells that are inherently capable of doing so. Interactions between fibroblasts and the collagenous extracellular matrix regulate type I procollagen synthesis. In sun-protected skin, collagen fibrils exist as a highly organized matrix. Fibroblasts are found within the matrix, in close apposition with collagen fibers. In photodamaged skin, collagen fibrils are shortened, thinned, and disorganized. The level of partially degraded collagen is approximately 3.6-fold greater in photodamaged skin than in sun-protected skin, and some fibroblasts are surrounded by debris. To model this situation, skin fibroblasts were cultured in vitro on intact collagen or on collagen that had been partially degraded by exposure to collagenolytic enzymes. Collagen that had been partially degraded by exposure to collagenolytic enzymes from either bacteria or human skin underwent contraction in the presence of dermal fibroblasts, whereas intact collagen did not. Fibroblasts cultured on collagen that had been exposed to either source of collagenolytic enzyme demonstrated reduced proliferative capacity (22 and 17% reduction on collagen degraded by bacterial collagenase or human skin collagenase, respectively) and synthesized less type I procollagen (36 and 88% reduction, respectively, on a per cell basis). Taken together, these findings indicate that 1) fibroblasts from photoaged and sun-protected skin are similar in their capacities for growth and type I procollagen production; and 2) the accumulation of partially degraded collagen observed in photodamaged skin may inhibit, by an as yet unidentified mechanism, type I procollagen synthesis.

Collagenolytic and gelatinolytic matrix metalloproteinases and their inhibitors in basal cell carcinoma of skin: comparison with normal skin.

Tissue from 54 histologically-identified basal cell carcinomas of the skin was obtained at surgery and assayed using a combination of functional and immunochemical procedures for matrix metalloproteinases (MMPs) with collagenolytic activity and for MMPs with gelatinolytic activity. Collagenolytic enzymes included MMP-1 (interstitial collagenase), MMP-8 (neutrophil collagenase) and MMP-13 (collagenase-3). Gelatinolytic enzymes included MMP-2 (72-kDa gelatinase A/type IV collagenase) and MMP-9 (92-kDa gelatinase B/type IV collagenase). Inhibitors of MMP activity including tissue inhibitor of metalloproteinases-1 and -2 (TIMP-1 and TIMP-2) were also assessed. All three collagenases and both gelatinases were detected immunochemically. MMP-1 appeared to be responsible for most of the functional collagenolytic activity while gelatinolytic activity reflected both MMP-2 and MMP-9. MMP inhibitor activity was also present, and appeared, based on immunochemical procedures, to reflect the presence of TIMP-1 but not TIMP-2. As a group, tumours identified as having aggressive-growth histologic patterns were not distinguishable from basal cell carcinomas with less aggressive-growth histologic patterns. In normal skin, the same MMPs were detected by immunochemical means. However, only low to undetectable levels of collagenolytic and gelatinolytic activities were present. In contrast, MMP inhibitor activity was comparable to that seen in tumour tissue. In previous studies we have shown that exposure of normal skin to epidermal growth factor in organ culture induces MMP up-regulation and activation. This treatment concomitantly induces stromal invasion by the epithelium (Varani et al (1995) Am J Pathol 146: 210-217; Zeigler et al (1996b) Invasion Metastasis 16: 11-18). Taken together with these previous data, the present findings allow us to conclude that the same profile of MMP/MMP inhibitors that is associated with stromal invasion in the organ culture model is expressed endogenously in basal cell carcinomas of skin.

Elaboration of matrix metalloproteinase inhibitors by human skin in organ culture and by skin cells in monolayer culture: relationship to invasion.

Functional and immunochemical approaches were used to assess matrix metalloproteinase (MMP) inhibitors, e.g., tissue inhibitor of metalloproteinases 1 and 2 (TIMP-1 and TIMP-2), in organ cultures of normal human skin maintained under growth factor free conditions or in medium supplemented with a combination of growth factors including epidermal growth factor, insulin, and pituitary extract. It has previously been shown that under growth factor free conditions, normal skin structure and function are maintained for several days, while in the presence of these exogenous growth factors, the epithelial cells invade the stroma [Invasion and Metastasis 1993;13:225-233]. TIMP-1 was detected in equivalent amounts in organ culture fluids under both conditions. TIMP-2 was not detected under either condition. Normal epidermal keratinocytes, normal dermal fibroblasts, and three different epithelial tumor cell lines were also examined for MMP inhibitor expression. Keratinocytes and fibroblasts produced high levels of both TIMP-1 and TIMP-2, but in neither cell type was there a significant difference between growth factor free and growth factor containing conditions. In contrast, the three epithelial tumor cell lines produced low to undetectable levels of both TIMP-1 and TIMP-2. These data suggest that acquisition of local invasive capacity is not dependent on a reduction in MMP inhibitor expression. A reduction in MMP inhibitors may accompany the transition from invasive to metastatic tumors.

Human squamous carcinoma cell invasion in organ-cultured skin.

In a previous study we showed that normal human epidermal keratinocytes were capable of invading the dermis of organ cultured skin when the tissue was treated with an exogenous source of epithelial growth factors (Fligiel and Varani (1993) Invasion Metastasis, 13, 225-233). Here we examined human squamous carcinoma cells from three different tumors for ability to invade the dermis in the same model. Invasion occurred in 40-80% of the tissues, depending on the tumor line, and was observed with equal frequency under both growth factor-free conditions and in the presence of exogenous growth factors. These data demonstrate that malignant human epithelial cells have the capacity to invade the dermis of organ-cultured skin. Unlike normal keratinocytes, there appears to be no exogenous growth factor requirement for invasion by the malignant cells.

All-trans retinoic acid inhibits fluctuations in intracellular Ca2+ resulting from changes in extracellular Ca2+.

Previous studies have shown that all-trans retinoic acid (RA) preserves fibroblast viability and stimulates their proliferation, in part, by reducing the extracellular Ca2+ requirement (Am J Pathol 1990, 130:1275). Based on this observation, we have in the present study examined the effects of RA on Ca2+ mobilization in human dermal fibroblasts. For these studies we used the Ca(2+)-binding dyes, Fluo-3 and Indo-1. Using fluorescence of Fluo-3-loaded cells or Indo-1-loaded cells as indicators of intracellular free Ca2+, we observed that treatment of the cells with RA did no, by itself, alter the concentration of intracellular Ca2+. Nor did it interfere with the rapid, transient rise in intracellular Ca2+ induced by treatment with ionomycin. However, treatment of the cells with RA prevented re-equilibration of intracellular Ca2+ when the cells were initially equilibrated in low Ca2+ (0.15 mmol/L) culture medium and then switched to high Ca2+ (1.4 mmol/L) medium or when cells were first equilibrated in high Ca2+ medium and then switched to low Ca2+ medium. This effect of RA could be seen within seconds after treatment and the effect was observed 1 day after treatment (longest time point examined). The effect was concentration dependent and concentrations of RA that modulated Ca2+ re-equilibration (0.3 to 3.0 mumol/L) were the same as those that have previously been shown to promote fibroblast survival and growth. A biologically inactive retinoid did not have this effect. Specificity of the response was suggested by the finding that concentrations of RA that modulated Ca2+ movement had no effect on Ba2+ transport. These data suggest that RA prevents re-equilibration of intracellular Ca2+ in human dermal fibroblasts by interfering with Ca2+ movement across the plasma membrane.

Human skin in organ culture. Elaboration of proteolytic enzymes in the presence and absence of exogenous growth factors.

Proteinase levels were assessed in organ culture fluids from human neonatal foreskin maintained under growth factor-free conditions and in the presence of a combination of growth factors (ie, epidermal growth factor, insulin, hydrocortisone, pituitary extract, and all-trans-retinoic acid). Analysis of culture fluids by gelatin zymography revealed the presence of 92-kd and 72-kd gelatinases. There was a greater amount of 92-kd gelatinase activity in the presence of growth factors whereas the levels of 72-kd gelatinase were similar in growth factor-free and growth factor-containing media. Experiments with keratinocytes and fibroblasts in monolayer culture and with isolated dermal tissue in organ culture indicated that the epithelial component was responsible for most of the 92-kd gelatinase activity whereas fibroblasts were primarily responsible for the 72-kd gelatinase activity. Activation with aminophenyl mercuric acetate, requirement for divalent cations, inhibition with EDTA, and insensitivity to inhibition with phenylmethyl sulfonyl fluoride indicated that both gelatinases were metalloproteinases. In additional studies, culture fluids were examined for the presence of plasminogen activator activity. This was detected in culture fluids from tissues maintained under both conditions but was increased in the growth factor-containing medium. The increased amount seen in the growth factor-containing medium appeared to be due almost entirely to a single factor, ie, all-trans-retinoic acid. In monolayer culture, both keratinocytes and fibroblasts produced plasminogen activator; the level was higher in keratinocyte culture fluids than in culture fluids from fibroblasts.

All-trans retinoic acid (RA) stimulates events in organ-cultured human skin that underlie repair. Adult skin from sun-protected and sun-exposed sites responds in an identical manner to RA while neonatal foreskin responds differently.

Adult human skin from a sun-protected site (hip) and from a sun-exposed site (forearm) was maintained in organ culture for 12 d in the presence of a serum-free, growth factor-free basal medium. Cultures were incubated under conditions optimized for keratinocyte growth (i.e., in 0.15 mM extracellular Ca2+) or for fibroblast growth (i.e., in 1.4 mM extracellular Ca2+). Treatment with all-trans retinoic acid (RA) induced histological changes in the organ-cultured skin under both conditions which were similar to the changes seen in intact skin after topical application. These included expansion of the viable portion of the epidermis and activation of cells in the dermis. In sun-damaged skin samples, which were characterized by destruction of normal connective tissue elements and presence of thick, dark-staining elastotic fibers, a zone of healthy connective tissue could be seen immediately below the dermo-epidermal junction. This zone was more prominent in RA-treated organ cultures than in matched controls. Associated with these histological changes was an increase in overall protein and extracellular matrix synthesis. In concomitant studies, it was found that RA treatment enhanced survival and proliferation of adult keratinocytes and adult dermal fibroblasts under both low- and high-Ca2+ conditions. In all of these assays, responses of sun-protected and sun-exposed skin were identical. In contrast, responses of neonatal foreskin to RA were similar to those of adult skin in the presence of low-Ca2+ culture medium, but under conditions of high extracellular Ca2+ RA provided little or no additional stimulus. Together these studies suggest that the ability of RA to enhance repair of sun-damaged skin (documented in previous studies) may reflect its ability to influence the behavior of skin in a manner that is age dependent but independent of sun-exposure status.

all-trans-retinoic acid preserves viability of fibroblasts and keratinocytes in full-thickness human skin and fibroblasts in isolated dermis in organ culture.

Human dermal fibroblast and human epidermal keratinocyte survival was examined under various conditions in organ culture. Using cell recovery from organ-cultured tissue as the criterion, it was observed that no keratinocytes and few fibroblasts survived incubation for 10-12 days in serum-free basal medium containing a low level (0.15 mM) of extracellular Ca2+. Increasing the extracellular Ca2+ concentration to 1.4 mM or treating the tissue with 3 microM retinoic acid (RA) under low Ca2+ conditions resulted in increased keratinocyte and fibroblast survival; the two treatments together were more effective than either treatment alone. The same treatments preserved fibroblast survival when pieces of isolated dermal tissue were incubated in organ culture and also supported fibroblast survival in monolayer culture. These findings indicate that recovery of keratinocytes and fibroblasts from skin after maintenance in organ culture provides a simple but definitive measure of the viability of the major cellular elements present in the tissue. These findings suggest that RA treatment enhances survival of both fibroblasts and keratinocytes and that these effects of RA can be seen at physiological Ca2+ concentrations as well as at suboptimal levels of extracellular Ca2+. Finally, these results indicate that the dermis is a direct target of RA.

Retinoid toxicity for fibroblasts and epithelial cells is separable from growth promoting activity.

Three different retinoids with widely varying capacity to stimulate skin repair in vivo and stimulate fibroblast and epithelial cell growth in vitro were examined for capacity to lyse the same cells at high concentrations. These included all-trans retinoic acid (RA), tetrahydro tetramethyl napthalenyl benzoic acid (TTNPB), and its biologically inactive structural analogue, meta-carboxy TTNPB. Despite their differing capacities to stimulate skin repair and cell growth, all of the agents were cytotoxic for fibroblasts and epithelial cells over the same range of concentrations (0.6-3 x 10(-5) M). Cytotoxicity for both fibroblasts and epithelial cells was blocked by addition of phosphatidylcholine to the cells along with the retinoid. In the presence of high concentrations of RA (0.75-3 x 10(-5) M) and phosphatidylcholine, proliferation was observed. The proliferative response was greater under these conditions than in the presence of an optimal concentration of RA (0.75-3 x 10(-6) M) without phosphatidylcholine. These data suggest that toxicity of retinoids can be separated, at least partially, from their growth-promoting activities.

All-trans retinoic acid and extracellular Ca2+ differentially influence extracellular matrix production by human skin in organ culture.

Two-mm full-thickness punch biopsies of human skin were placed in organ culture in a serum-free, growth factor-free basal medium. Under conditions of low extracellular Ca2+ (0.15 mmol/L), the tissue quickly degenerated. However, degeneration was prevented when the extracellular Ca2+ concentration was increased to 1.4 mmol/L. The tissue remained histologically normal in appearance and biochemically active for up to 12 days. The addition of 3 mumol/L all-trans retinoic acid (RA) to the low-Ca2+ culture medium also prevented tissue degeneration. However, in contrast to what was seen in the presence of 1.4 mmol/L Ca2+, epidermal differentiation did not occur normally in the presence of RA. Rather, the upper layers of the epidermis routinely separated from the underlying basal cells. Fibronectin production by the organ cultured skin was examined. Biosynthetic labeling/immunoprecipitation studies demonstrated that incubation of the tissue in basal medium containing 1.4 mmol/L Ca2+ resulted in a high level of fibronectin production relative to the amount produced in basal medium containing 0.15 mmol/L Ca2+. In contrast, the addition of 3 mumol/L RA to the low Ca2+ basal medium did not stimulate fibronectin production. Similar results were observed in enzyme-linked immunosorbent assays where the addition of Ca2+ to a final concentration of 1.4 mmol/L stimulated fibronectin and thrombospondin production whereas RA (3 mumol/L) did not. Although RA by itself failed to stimulate extracellular matrix production, the addition of 3 mumol/L RA to basal medium containing 1.4 mmol/L Ca2+ led to a further increase in fibronectin production over that seen in the presence of 1.4 mmol/L Ca2+ alone. Taken together, these data indicate that although either 1.4 mmol/L Ca2+ or 3 mumol/L RA facilitates survival of organ-cultured skin in basal medium, they have very different effects on extracellular matrix production. This supports the view, based on histological appearance, that the two treatments work through different mechanisms. The data further support the suggestion that the two treatments may have additive or even synergistic effects.

Effects of all-trans retinoic acid and Ca++ on human skin in organ culture.

In this study, we have established an organ culture model of human skin and examined the effects of both all-trans retinoic acid (RA) and extracellular Ca++ on the epidermal and dermal components of the organ-cultured skin. Our data show that while organ cultures maintained in serum-free, growth factor-free culture medium containing 0.15 mM Ca++ degenerated rapidly, those treated with concentrations of RA that have been shown previously to stimulate fibroblast and keratinocyte proliferation in monolayer culture (J Invest Dermatol 1989, 93:449; 1990, 94:717; Am J Pathol 1990, 136:1275) demonstrated a healthy appearance for up to 12 days. Degeneration of the control cultures was characterized by separation of the epidermis from the underlying dermis, progressive cell necrosis leading to a complete absence of viable cells from both the dermal and epidermal compartments, disintegration and fibrillation of the dermal connective tissue, and a cessation of protein synthesis. RA-treated organ cultures contained large numbers of healthy-appearing cells in both the epidermal and dermal compartments. One or several layers of viable basal cells in the epidermis could be seen at least through day 12. However, the upper layers of the epidermis frequently separated from the cells in the basal layer. The dermal connective tissue was histologically well-preserved. Furthermore, the level of protein synthesis was higher in the RA-treated cultures than in the control cultures. In addition to treating organ cultures with RA, other cultures were exposed to serum-free, growth factor-free culture medium containing 1.4 mM Ca++. The presence of the elevated Ca++ concentration also preserved cellular and connective tissue structures in the dermal and epidermal compartments. In comparison to RA there was better preservation of the overall epidermal structure. The upper layers of epidermal cells did not separate from the basal cells, and the various stages of epithelial differentiation could be seen. Histologically, the dermis was well-preserved in the presence of elevated extracellular Ca++. Specimens treated with a combination of Ca++ and RA demonstrated features consistent with the features induced by each treatment separately. This included an expanded basal layer of epithelial cells and a prominent keratotic layer with a fairly orderly pattern of differentiation. The tendency of the upper epidermis to separate from the basal cells was partially mitigated. Taken together, these data indicate that both RA and extracellular Ca++ act to prevent the degeneration of human skin in organ culture but probably do so through different mechanisms.

Effects of sodium lauryl sulfate on human skin in organ culture: comparison with all-trans-retinoic acid and epidermal growth factor.

Human skin organ cultures were established from 2-mm punch biopsies and incubated under serum-free conditions in basal medium containing either 0.15 or 1.4 mM extracellular Ca2+. Organ cultures were treated with concentrations of sodium lauryl sulfate (SLS) that had previously been shown to support growth of human epidermal keratinocytes and human dermal fibroblasts in monolayer culture. Epidermal growth factor (EGF), alone and in combination with insulin and bovine pituitary extract, fetal bovine serum and all-trans retinoic acid (RA) were also examined for comparative purposes. The addition of SLS to culture medium containing low extracellular Ca2+ had no effect on the viability or histological appearance of the organ-cultured skin. Complete degeneration of the tissue occurred in the presence of SLS just as it did under control conditions. When SLS was added to culture medium containing high extracellular Ca2+, the basal layer of keratinocytes was much thinner than under control conditions. When EGF or EGF in combination with insulin and pituitary extract were utilized in place of SLS, identical results were obtained. That is, there was no preservation of the basal epithelial layer in the presence of low-Ca2+ culture medium and in the presence of high-Ca2+ culture medium, the basal layer was thinner than in control tissue. Virtually identical results were also obtained in medium containing 10% fetal bovine serum. In contrast, when RA was included in low-Ca2+ culture medium, the basal epithelium was maintained in a viable, histologically healthy condition. However, normal epithelial differentiation did not occur and the upper layers of the epidermis separated from the basal cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Arachidonic acid metabolism in murine fibrosarcoma cells with differing in vivo and in vitro characteristics.

Arachidonic acid metabolism was examined in a series of strongly malignant murine fibrosarcoma cell lines and in a series of weakly malignant lines isolated from the same tumors. The cells were examined in the unstimulated state and after stimulation with 12-O-tetradecanoyl phorbol acetate (TPA), laminin or fibronectin. All 3 agents were known from previous studies to induce adherence and motility in the murine fibrosarcoma cells. When the cells were prelabelled with 3H-arachidonic acid, all 3 agents stimulated the release of radioactivity into the supernatant fluids. The response to TPA was rapid while the response was slower but sustained when either laminin or fibronectin was used as the stimulating agent. This is of interest because TPA induces a rapid but transient adherence response in the same cells while laminin and fibronectin induce a slow, sustained response. Examination by radioimmunoassay procedures indicated that both control cells and stimulated cells were able to produce a variety of lipoxygenase and cyclooxygenase metabolites. In quantitative terms, the strongly malignant cells were more active than their weakly malignant counterparts. They released greater amounts of radioactivity into the supernatant fluid and produced a greater quantity of arachidonic acid metabolites, particularly prostaglandin E2, than did the corresponding weakly malignant cells. This is of interest because previous studies have shown that while both the strongly and weakly malignant cells respond in the adherence assay to TPA, laminin and fibronectin, only the strongly malignant cells demonstrate directional motility (chemotaxis and haptotaxis).

Directional motility in strongly malignant murine tumor cells.

Laminin, fibronectin and 12-O-tetradecanoyl phorbol acetate (TPA) were examined for their ability to induce biological responses (cell-to-substrate adherence and motility) in a series of strongly malignant and weakly malignant murine fibrosarcoma cells. All three agents caused increased cell-to-substrate attachment of the cell lines. Laminin and fibronectin induced a slow but sustained response, whereas with TPA the response was rapid and transient. The 3 agents also stimulated motility in the same cells. Random motility was seen with all of the cells but directional motility was observed primarily in the strongly malignant cells. With TPA, the response was chemotactic but laminin and fibronectin induced cell migration by haptotaxis. Since laminin and fibronectin are constituents of the extracellular matrix and since malignant tumor cells must cross the extracellular space during invasion, those properties of cells which allow for migration across a substrate containing these materials may contribute to the expression of malignancy.

Response of Walker 256 carcinosarcoma cells to 12-O-tetradecanoylphorbol 13-acetate: possible regulation by endogenous cyclooxygenase metabolites.

Walker 256 carcinosarcoma cells (Walker cells) maintained in suspension culture responded to stimulation with 12-O-tetradecanoylphorbol 13-acetate [(TPA) CAS: 16561-29-8] by becoming temporarily adherent to the substratum. Both the control and treated cells produced very low levels of cyclooxygenase metabolites as detected by radioimmunoassay procedures. Levels of prostaglandin F2 alpha, 6-keto prostaglandin F1 alpha (6-keto PGF1 alpha) (a prostacyclin metabolite), and thromboxane B2 were virtually the same as background, and prostaglandin E2 (PGE2) levels were only slightly higher. Studies employing high-performance liquid chromatography also failed to detect significant quantities of cyclooxygenase products in the supernatants from either the control or the stimulated Walker cells. Although the Walker cells maintained in culture failed to produce significant amounts of cyclooxygenase metabolites, they produced much greater amounts of these products, particularly PGE2 and 6-keto PGF1 alpha when they were maintained as an ascites tumor. Concomitant with the production of these metabolites was a loss in responsiveness to TPA in the adherence assay. Upon reestablishment in culture, the cells gradually reacquired the ability to respond to TPA. Over the same period, synthesis of cyclooxygenase products was curtailed. If the cells taken from ascites tumors were incubated with indomethacin so as to inhibit the production of cyclooxygenase metabolites, they rapidly regained responsiveness to TPA. These findings suggest that stimulus-coupled responses in the Walker cells may be regulated, at least in part, through the production of endogenous cyclooxygenase metabolites.