Sean F. Mullins

Ozone in the upper atmosphere protects us against the harmful UV radiation from the sun. Our atmospheric defenses against UV radiation have begun to erode since the 1960’s. In 1985, a hole in the ozone layer above Antarctica was first discovered. Since then, the hole has gotten bigger and another hole has appeared over the Arctic Circle. Loss of ozone has been observed over the U.K and the U.S. Loss of ozone leads to elevated levels of UV radiation, especially UV-B, striking the surface of the earth. A detailed discussion of the theories behind the development of the ozone hole is beyond this paper, but I will discuss some of the effects of this depletion. Specifically, I will focus on how UV radiation affects cells.

Ozone depletion is believed to be caused by clorofluorocarbons (C.F.C’s) that are released into the atmosphere. These CFC’s react with ozone and destroy it. In April of 1992, the United Nations Environmental Program (UNEP) presented a report that said a 1% loss of ozone would produce 100,000 to 150,000 additional cases of cataract induced blindness worldwide. The UNEP has ruled that UV-B radiation is a threat to human health, crops and marine ecology. In April 1991, the EPA reported that in the next 50 years, there will be an estimated 12 million new cases of skin cancer and 200,000 people will die from malignant melanoma as a result of UV-B radiation reaching the surface of the earth.

What exactly does UV light do to cells? UV radiation can cause apoptosis and cancer in mammalian cells (Wang, 1999). UV light can also cause clustering and internalization of the cell surface receptors for epidermal growth factor (EGF), tumor necrosis factor (TNF) and interleukin 1 (IL1). (Wang, 1999). The jun n-terminal kinase (JNK), also known as stress activated protein kinase (SAPK), pathway is known to respond to extracellular stimuli that induce apoptosis such as tumor necrosis factor-a, ceramide, granzyme B, interleukin-1, UV light and g-radiation (Frisch, 1996). Early responses to UV radiation include activation of the transcription factors for c-fos and c-jun genes (Wang, 1999). These two gene products bind together to form the fos-jun complex, also known as AP-1 (Zubay, 1998). The fos and jun proteins belong to a class of DNA binding proteins known as leucine zippers (Zubay, 1998). Normally, AP-1 is activated by protein kinase C, which is activated by a signal from the cell surface (Zubay, 1998). AP-1 can lead to either cell division, or apoptosis, thus an AP-1 that has gone haywire could lead to tumorigenesis (Zubay, 1998). UV radiation also leads to the activation of two mitogen activated protein kinase pathways, JNK/SAPK, and p38 (Wang, et al. 1999). In normal cells, a mitogenic effector, such as epidermal growth factor, binds to a mitogenic receptor, such as epidermal growth factor receptor. This binding activates the Ras GTP binding protein, which activates the protein Raf-1. Raf-1 phosphoryates mitogen activated protein kinase kinase (MEK). MEK phosphorylates mitogen activated protein kinase (MAPK) which leads to cell differentiation and mitosis. In response to stress, Ras GTP activates MEKK, (MAPK kinase kinase). MEKK phosphorylates stress activated protein kinase kinase (SEK). SEK phosphorylates stress activated protein kinase (SAPK) which causes the proteins c-jun and c-fos to be produced. C-fos and c-jun form AP-1, which binds DNA and can lead to apoptosis (Zubay, 1998; Sanchez, 1994; Yan, 1994).

Cells that die by apoptosis degrade their DNA and begin to shrink a stark contrast to swelling and eventual bursting of cells that die by necrosis (Wang, 1999). The decrease in cell volume in apoptitic cells is mediated by the loss of K⁺ and Na⁺ (Bortner, 1997). Studies have suggested that the efflux of K⁺ and Na⁺ can trigger apoptosis (Bortner, 1997). The purpose of this study by Bortner was to see if there was a relationship between K⁺ channel activity and UV induced apoptosis (Wang, 1999).

The K⁺ channel is a membrane spanning protein that has six a-helical membrane spanning regions. The protein has a voltage sensor domain and a domain with negatively charged amino acid residues. There are three conformations in the K⁺ channel, closed, open and inactive. The channels are voltage dependent and the exact molecular mechanism of the voltage gated control remains to be discovered. The Na⁺ and K⁺ channels are separate but similar. Unicellular eukaryotes such as Paramecium and bacteria have voltage gated K⁺ channels. The absence of Na⁺ channels in these organisms suggests a common K⁺ channel ancestor and organisms with Na⁺ channels evolved later. Spectroscopic studies have provided evidence of how the channel is turned on and off. A ball and chain type motif is used. The ball is positively charged and can swing and attach to the base of the channel, which is negatively charged. This attachment closes the channel by physically preventing ions from passing into or out of the cell. The channel is open or active when the ball swings away from the opening.

We already know that UV light can disrupt chemical bonds in the DNA with the formation of thymine dimers (Zubay, 1998). Therefore, it is reasonable to assume that U.V light disrupts enough bonds in the K⁺ channel that it effectively destroys the protein.

Patch clamp recording is the main method of studying ion movement and ion channels in living cells. It is possible to record the activity of a single ion channel. A fine glass tube called a microelectrode or a patch pipette is attached to the membrane of a cell or part of a membrane. The cell attached patch clamp was used in this experiment to detect K⁺ channel activity. The tip of the patch pipette is only a few micrometers in diameter. The tip is filled with an aqueous conducting solution and the tip of the patch pipette is attached to the cell via gentle suction. A metal recording wire is then inserted in the large end of the microelectrode and attached to an oscilloscope. A second electrode is placed in a bath with the cell. To test which ions go through the channel; the concentration of ions in the medium on either side of the patch can be varied. The voltage across the membrane patch, the membrane potential, can be held constant. How changes in membrane potential effect the opening and closing of the channels can be determined (Alberts, 1998).

Peaks on the oscilloscope indicate when and how strongly the channels open. In the work done by Wang, the patch pipettes contained 140mM KCl, 0.05mM CaCl₂, 2mM ATP, 0.05mM GTP, 1mM EDTA, and 200mg/ml nystatin and were maintained at pH 7.2. The bath solution was composed of 140mM NaCl, 2mM MgCl₂, and 0.05mM CaCl₂.  The cell attached patch clamp was used to measure the response of the K⁺ channels to UV-B light.

Myeloblastic leukemia cells were obtained from a human patient and were cultured in a medium called a Roswell Park Memorial Institute (RPMI) 1640 culture medium containing 7.5% heat inactivated fetal bovine serum and 25mM of HEPES buffer. The culture was incubated at 37C. Patch clamp experiments were used to measure a single membrane channel activity.

Results of the patch clamp recording indicate that UV radiation stimulates K⁺ channel activity. The activity of the channel increased almost seven times within thirty seconds of exposure to the UV light. A K⁺ channel inhibitor (4-AP) prevented activation of the K⁺ channel when exposed to UV light. Patch pipettes that were filled with 100mM 4-AP showed no change in K⁺ channel activity. Cells that were exposed to 4-AP and UV light did not go into apoptosis. The effect of 4-AP is a time dependant process. 4-AP has a protecting effect for the cell against UV radiation. When 4-AP was added five seconds after the onset of UV radiation, 30% of the cells died by apoptosis. When 4-AP was added before or at the onset of UV radiation, it completely prevented cell death. These results indicate that an early effect of UV irradiation is the direct stimulation of K⁺ channels (Wang, 1999).

It has been shown that UV light and osmotic stress activate the JNK signaling pathway in many cells (Rossette, 1996). To see if there is a link between K⁺ channel activity and the JNK signaling pathway, 4-AP was added and the myeloblastic leukemia cells were exposed to UV light. Measuring the JNK activity by Western blotting would show if there was a link. If there was activity, then there is no link, or a weak one. If there was no activity, then there is a clear link. There was almost no activity in the JNK-1 when exposed to UV light, this indicates that the K⁺ channel activation is linked to the JNK (Wang, 1999).

There are two ways that apoptosis can be detected. One of the characteristics of apoptosis is DNA fragmentation. The basic idea is to extract DNA from cells and to run it on a 1.5-% agarose gel. A long smear will develop on the gel when it is stained with ethidium bromide. This indicates DNA fragmentation and apoptosis. A normal cell would have one dense band at the top of the gel. DNA was extracted from the cells by first washing the cells with a phosphate buffered saline solution. Next, a lysis buffer containing 200mM pH 8 Tris-HCl, 100mM EDTA, 1% SDS, and 100mg/ml proteinase kinase was added. The cells were then incubated at 55 C for four hours (Wang, 1999).

Tris buffers the DNA solution. EDTA protects the DNA from DNAases by binding divalent cations that are necessary cofactors for DNase activity. SDS is a detergent that dissolves the cell membrane and other cellular components. DNA is precipitated out of the lysate with ethanol or isopropanol (Micklos, 1990).

Apoptosis can also be detected by using a fluorescence microscope. The cells can be stained with ethidium bromide and acridine orange. Under the fluorescent microscope, the nuclei that have stained green have not lost their membrane integrity, but those staining orange have lost their membrane integrity. Loss of membrane integrity is another indicator of apoptosis (Wang, 1999).

Cells were exposed to etoposide (an apoptosis inducer) and 4-AP. The etoposide inhibits topoisomerase II in the nucleus, and thus kills the cell. The presence of 4-AP had no effect on etoposide induced cell death. These results show that K⁺ channel hyperactivity is not linked to the etoposide mechanism (Wang, 1999).

Like JNK, p38 is also a stress activated mitogen activated protein kinase and it is activated in response to UV radiation. The activity of p38 was measured, by Western blotting, with and without 4-AP to see if the p38 response is mediated by K⁺ channel activity. P38 was active regardless of the presence of 4-AP. The lack of effect by the K⁺ channel inhibitor shows that p38 activation by UV light is independent of K⁺ channel activity. The exact mechanism of p38 activation remains to be determined (Wang et al. 1999).

Western blotting was used to measure the activity of stress activated protein kinase kinase (SEK) and stress activated protein kinase (SAPK/JNK). This was done to prove that K⁺ channel activation is the first step in the SAPK/JNK pathway to apoptosis (Wang, 1999). Western blotting is similar to Southern blotting except that it assays for proteins instead of DNA. The procedure is to first isolate the proteins of interest using SDS poly-acrylamide gel electrophoresis. The gel is then placed on a polyvinylidene diflouride membrane (Millipore) and a transfer buffer is used to force the proteins out of the gel and onto the membrane. Radio labeled antibodies that are specific for the proteins in question are then added to the membrane. The antibodies bind to the protein and can now be visualized by autoradiography (Elseth and Baumgardner, 1996; Wang, 1999). Western blotting analysis showed that SEK was strongly activated by phosphorylation after being exposed to UV light. When the K⁺ channel blocker 4-AP was added, SEK was not activated. This indicates that suppression of UV induced K⁺ channel activity inhibits the early events in the cell membrane. These early events precede JNK activation. This also indicates that K⁺ channel activation is the first step in the cascade that leads to apoptosis (Wang, 1999).

Conclusions: patch clamp recording showed that K⁺ channel activity was stimulated by exposure to UV radiation. Western blotting showed that K⁺ channel activity caused by exposure to UV radiation activates the JNK/SAPK signaling pathway. The JNK/SAPK pathway does lead to apoptosis and cells exposed to UV radiation did die by apoptosis. These data prove that K⁺ channel hyperactivity caused by UV light is the first step in the JNK/SAPK signaling cascade that leads to apoptosis.

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