Thapsigargin Induces Rapid, Transient Growth Inhibition and c-fos Expression Followed by Sustained Growth Stimulation in Mouse Keratinocyte Cultures

Abstract

Although the sesquiterpene lactone thapsigargin has been shown to possess hyperplastic and tumor-promoting activities when applied topically to mouse skin in vivo, the cellular mechanism(s) which underlie these effects are unclear. We show here that thapsigargin treatment of Primary mouse epidermal keratinocytes increased intracellular free Ca2+ concentration (Cai) in a concentration-dependent manner. Thapsigargin induced a rapid, transient elevation in keratinocyte Cai, in part due to the release of Ca2+ from intracellular stores. This response was followed by a sustained elevation in Ca2+, resulting entirely from calcium influx. Thapsigargin elicited a biphasic effect on keratinocyte DNA synthesis: a rapid inhibitory effect (50-60% inhibition at 4-8 h), followed by a very marked and sustained elevation. Prolonged treatment of keratinocytes with thapsigargin at relatively high concentrations resulted in cytotoxicity (inhibition of neutral red uptake). The rapid antiproliferative effect of thapsigargin was not associated with cytotoxicity, as determined by either neutral red uptake or by trypan blue exclusion, and was not blocked by pretreatment with Ro 31-7349, a selective inhibitor of protein kinase C. The rapid antiproliferative effect of thapsigargin was associated with rapid, transient activation of keratinocyte c-fos expression and rapid inhibition of total protein synthesis. Taken together, these findings raise the possibility that the hyperplastic and tumor-promoting activities of thapsigargin on epidermis in vivo result from direct keratinocyte growth stimulation as a consequence of a prolonged elevation in levels of Cai.

Phytol Is a Novel Tumor Promoter on ICR Mouse Skin

“…34) It is of interest that Elmets et al 22) demonstrated a significant difference in rates of cutaneous tumorigenesis between different strains of mice and moreover, biphasic effects on cell proliferation and growth inhibition by tumor promoters, such as thapsigargin, 35) okadaic acid, 36) and TPA, 37) have been reported. Thus, it is likely that differences in dose, administration route or mouse strain influence tumorigenesis induced by phytol.…”

Haploinsufficiency of Atp2a2, Encoding the Sarco(endo)plasmic Reticulum Ca2+-ATPase Isoform 2 Ca2+ Pump, Predisposes Mice to Squamous Cell Tumors via a Novel Mode of Cancer Susceptibility

“…However, an alternative possibility is that the tumors arise in rare cells in which the loss of Atp2a2 heterozygosity has occurred, which would be similar to mechanisms involving known tumor suppressor genes. Thapsigargin, an irreversible inhibitor of SERCA2 activity, serves as a tumor promoter in multistage mouse skin carcinogenesis (10) and has been shown to induce DNA synthesis (11) and cause growth stimulation (12) in cultured keratinocytes. It is unclear, however, whether these effects are due to partial inhibition of SERCA2, which would resemble haploinsufficiency, to complete inhibition, which would resemble loss of heterozygosity (LOH), or to some other effect that is independent of SERCA2.…”

Thapsigargin-inducedgrp78 expression is mediated by the increase of cytosolic free calcium in 9L rat brain tumor cells

“…Moreover, because the release of Ca 2ϩ from intracellular storage pools and the entry of extracellular Ca 2ϩ are often coupled, treatment with TG, while blocking Ca 2ϩ accumulation and allowing Ca 2ϩ release from ER, induces a rapid increase of [Ca 2ϩ ] c followed by a sustained increase mediated by a specific Ca 2ϩ influx pathway [Ikari et al, 1997;Wei et al, 1998]. Depending on cell types and treatment protocols, exposure to TG may induce the expression of various genes, including the 78-kDa glucose-regulated protein (GRP78) in Chinese hamster ovary cells [Li et al, 1993] and human rhabdomyosarcoma cells [Delpino et al, 1998], stress proteins in mammalian chondrocytes [Cheng and Benton, 1994], immediate early gene VL30 [Magun and Rodland, 1995] and interleukin-6 in murine macrophages [Bost and Mason, 1995], zinc finger transcription factor, EGR-1, in human melanoma cells [Muthukkumar et al, 1995], c-fos in keratinocytes [Harmon et al, 1996], as well as the growth arrest gene, gadd153, in prostate cancer cells [Lin et al, 1997]. Recently, it has been suggested that TGinduced gene expression in nonexcitable cells is dependent on calcium influx [Rodland et al, 1997].…”