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| TITLE: | Ion Changes in Apoptosis: A possible mechanism for EMF effects | ||
| Principal Investigator |
Elizabeth Murphy, Ph.D. | Laboratory of Molecular Biophysics, NIEHS | |
| Health Relevance |
Cancer | ||
| Research Categories |
Cell Function | Cell Processes | Cell Proliferation |
| FY95 Funds | DIR-7 $ 60,000 | Start Date 9/93 | End Date 9/95 |
| Rationale and Summary |
Epidemiologic studies have suggested that exposure to electromagnetic fields (EMF) may cause an increased incidence of some forms of cancer; however, the mechanism responsible for this is unclear. The multistage process of neoplastic progression requires the uncoupling of proliferation and apoptosis in such a manner that the cancer cell acquires a proliferative advantage. Because EMF has been associated with leukemia, a disease associated with aberrant inhibition of apoptosis, an investigation of the effect of EMF on the regulation of apoptosis is particularly relevant. The mechanism of the EMF effect is under investigation, however, there are several reports suggesting that EMF can modulate Ca2+ homeostasis. We are testing the hypothesis that EMF exposure alters Ca2+ homeostasis, and that altered Ca2+ homeostasis alters signaling pathways that lead to a proliferative advantage of the preneoplastic cell, and thus enhances neoplastic progression. To test this, we employed three lines of Syrian hamster embryo (SHE) cells that represent different stages of neoplastic progression. Earlier studies showed that, unlike normal diploid SHE cells whose growth arrests upon removal of serum, early preneoplastic cells, sup+I, undergo apoptosis at a high rate (Preston et al). Cells from a later stage of progression, sup-II, isolated from the same clone at a later passage, were resistant to apoptosis. We investigated Ca2+ homeostasis in these cells, and how it is altered by low serum, which induces apoptosis in the sup+I cells. We found differences in intracellular free Ca2+ levels, and also differences in endoplasmic reticulum Ca2+ compartmentalization, among the three cell lines, implying that Ca2+ regulation is modified during neoplastic conversion. We found a decrease in endoplasmic reticulum calcium in cells that will undergo apoptosis. This decrease in endoplasmic reticulum calcium is strongly associated with apoptosis; if we rescue cells from apoptosis we block the decrease in endoplasmic reticulum calcium. Furthermore this decrease in endoplasmic reticulum calcium appears to be due to altered plasma membrane calcium transport. Because EMF has been reported to alter membrane calcium transport this is a possible mechanism by which EMF could promote neoplasmic progression. We plan on investigating whether EMF exposure alters basal intracellular free Ca2+ levels in the cells at different stage of neoplastic progression, and whether it alters the response of the cells to low serum, which induces apoptosis in the sup+I cells. In particular we will investigate whether EMF exposure will block the decrease in endoplasmic reticulum calcium and apoptosis in sup+I cells in low serum. | ||
| Experimental Design and Exposure Conditions |
Syrian hamster embryo (SHE) cell lines and two immortalized cell lines derived from these cells were employed. The cell lines consist of normal diploid SHE cells, which growth arrest upon removal of serum, preneoplastic sup+I cells, which undergo apoptosis in low serum, and sup- cells which are resistant to apoptosis in low serum. We studied calcium homeostasis using fluorescent microscopy in cells loaded with the fluorescent calcium indicator, fura-2. Endoplasmic reticulum calcium was measured using thapsigargin releasable calcium. During our first year of funding we have characterized calcium homeostasis in these three cell lines at different stages of neoplastic progression. In the next year we will repeat these studies in the FDA exposure chamber to determine the effects of EMF. | ||
| Quality Assurance Measures |
In the upcoming year, we plan on using the FDA exposure chamber. We will utilize the expertise at the NIEHS funded exposure facility. | ||
| Results and Discussion |
In our first year of funding we have made excellent progress in defining changes in Ca2+
homeostasis associated with apoptosis. Fluorescent microscopy of fura-2 loaded cells was used to
measure intracellular, free calcium concentrations ([Ca2+ ]I) and endoplasmic reticulum (ER) Ca2+
content. We found that preneoplastic sup+I cells, which exhibit a high susceptibility to apoptotic
death, have an alteration in Ca2+ homeostasis, the consequence of which is lower [Ca2+ ]I and a
deficiency in ER Ca2+ replenishing mechanisms. Later stage sup-II preneoplastic cells, which are
less susceptible to apoptotic death, have developed a mechanism to block ER depletion, and thus
ER Ca2+ stores can be sustained. We examined mechanisms that might be responsible for the
observed decrease in ER Ca2+. Our data are consistent with altered plasma membrane Ca2+
transport as being involved in the low ER Ca2+ in sup+I cells. Current research is directed toward
the postulation that EMF-induced alterations in Ca2+ homeostasis modify the regulation of
apoptosis, thereby conferring a proliferative advantage to cells undergoing neoplastic conversion.
These data will be used for a collaborative project between NIOSH, the Laboratory of Molecular Biophysics, NIEHS, and the Laboratory of Molecular Carcinogenesis, NIEHS to assess the effect of EMF on calcium homeostasis and cell cycle regulation. |
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| Recent Publications |
Preston GA, Barrett JC, Biermann JA, Murphy E. Decreased Endoplasmic Reticulum Ca 2+ Stores and Activation of Apoptosis. In preparation. | ||