p53+Mutations+in+Melanoma

toc

=Introduction = Jacey Nishiguchi and Michelle Fat

After introducing several key concepts, we examine the use of p53 mutations as a more quantitative measure for assessing the effectiveness of sunscreen.

Useful Terminology Quick and easy definitions of selected terms

=The Problem: Skin Cancer =

**What is Skin Cancer? **
Skin cancer is the most common cancer in the U.S., and is caused by mutations in the DNA that go unrepaired, and are normally a result of UV radiation from the sun or tanning beds. Types of skin cancer include basal cell carcinoma (BCC), melanoma, and squamous cell carcinoma (SCC). BCC results from abnormal growth in the deepest layer of the epidermis, but normally don’t metastasize. Melanoma is the most deadly type of skin cancer; malignant tumors form as a result of unrepaired, damaged DNA whose mutations are then replicated and spread. These tumors form in the basal layer of the epidermis. The last type of skin cancer is SCC, which results from abnormal cells in the upper layers of the epidermis.   **Figure 1.** Images of skin with each type of Skin Cancer. Source: http://www.skincancer.org/skin-cancer-information

**How are skin cancers caused? **
 Skin cancers are the result of DNA damage in the cells of the skin that usually develops in the epidermis, or the top layer of skin. Ultraviolet (UV) radiation from the sun is the number one cause of DNA damage to the skin and therefore skin cancer. Artificial forms of UV radiation found through sunlamps and tanning booths can cause skin cancer as well. UV radiation is one of the types of “lights” on the spectrum that we measure from the earth to the sun. Because its wavelengths are shorter than visible light, our naked eye cannot detect them. The two main classifications of UV are UVA and UVB, both of which enter through the atmosphere and come in contact with human skin. UV light can interact with the skin and can alter pyrimidines, disrupt gene linkages, or even have deletion effects with the genome. Though DNA repair mechanisms exist, mistakes are made and failures happen, both which can lead to apoptosis or proliferation of abnormal cells.

 UVA rays are accountable for about 95% of UV radiation experienced on Earth, as they penetrate through the air with equal intensity during all hours of light and all seasons of the year. UVA can pass through glass and still have the same effect. These rays reach the dermis of human skin, contributing to aging, wrinkling, and the development of melanoma. UVA is the prime emission of tanning beds, as its exposure to the skin causes the skin to darken in an attempt to prevent further DNA damage.

 UVB rays are the main reason we get sunburnt and red after a long day outside, contributing to about 5% of UV radiation experienced on Earth. These rays do not penetrate as deep into the skin, mainly affecting the epidermal layers and playing a key role in the development of melanoma tumors. Unlike UVA, the intensity of UVB is dependent on time of day, season, and location on Earth. UVB is also unable to pass through glass and have a significant effect.

=Sunscreen =

**How can we protect against skin cancer? **
<span style="font-family: 'Times New Roman',Times,serif;"> One of the most common practices used to protect against skin cancer is the application of sunscreens to the skin.

<span style="font-family: 'Times New Roman',Times,serif;">What is sunscreen?
<span style="font-family: 'Times New Roman',Times,serif;"> Sun exposure is inevitable, as is resulting DNA damage. In an attempt to protect our skin from damaging effects, humans have come up with some sort of sun “screen” we are all told to put on without real knowledge about its protecting mechanism. So what is sunscreen? Sunscreens are products that consist of a combination of ingredients that help to prevent the skin from ultraviolet (UV) radiation. They are topical solutions that contain molecular compounds that can absorb, reflect, or scatter UV photons.

<span style="font-family: 'Times New Roman',Times,serif;">SPF
<span style="font-family: 'Times New Roman',Times,serif;"> SPF, or Sun Protection Factor, is not only a basic idea that you must understand before reading our final project, but also is a concept that is relevant to our day-to-day lives. So what is it? SPF is a mean of classifying the degree of protection that sunscreen provides against solar radiation, an in vivo assessment that offers quantitative results. According to the Skin Cancer Foundation, SPF 15 prevents reddening of the skin for 15 times longer than one would experience with unprotected skin. SPF 15 filters out about 93% of incoming UVB rays, while SPF 30 filters about 97% and SPF 50 98%. But what is the definition of “reddening” and what is happening on a molecular level with our skin and its interaction with UV light? Is a red color on our skin the first indicator of having skin cancer potential or is there damage occurring prior to any visible change? This unreliability causes us to believe there may be a more consistent and accurate indicator of DNA damage and possible skin cancer.

<span style="font-family: 'Times New Roman',Times,serif;">How is the effectiveness of sunscreen traditionally measured?
<span style="font-family: 'Times New Roman',Times,serif;"> Excessive sun exposure causes skin damage recognizable through erythema, edema, hyperplasia, the formation of sunburn cells, photoaging, immunosuppression, and skin cancer. While commercial sunscreens were originally developed to protect against erythema, recent research has studied the possibility that certain sunscreens efficiently protect against these effects of skin damage as well. Past researchers have measured the effectiveness of sunscreen by evaluating formulas based on their ability to prevent erythema caused by the exposure of human skin to UV radiation. The authors we followed acknowledge that there could be a fault in this way of thinking. The exact relationship between erythema and skin cancer is unknown and there may be more accurate predictors of sunscreen effectiveness.

<span style="font-family: 'Times New Roman',Times,serif;"> With this in mind, the researchers assessed if a mutation in the p53 tumor suppressor gene serves as a “short-term molecular surrogate for the long-term biologic end-point of skin cancer development”. In an attempt to develop earlier end-points for skin cancer indication, ones more accurate and timely than erythema, researchers set up experiments that challenged the traditional approaches.

=<span style="font-family: 'Times New Roman',Times,serif;">The p53 Gene: A Better Indicator =

<span style="font-family: 'Times New Roman',Times,serif;">What is the p53 Gene?
<span style="font-family: 'Times New Roman',Times,serif;"> The [|p53 gene] is a tumor suppressor gene, known to be responsible for about 100 proteins and whose function is to maintain the normal growth and stability of the cell cycle through either arresting the cell in G1, inducing apoptosis, DNA repair, or by inhibiting angiogenesis and metastasis. By arresting the cell in G1, the cell is able to repair and remove possible mutations before it can go into S phase and M phase. Basically, if the p53 gene is mutated, it won't produce the p53 protein and the cell loses its ability to repair itself, which can result in proliferation of mutated DNA. Studies have shown that because the p53 gene is related to regulation of the cell and management of mutations and repair, it can also serve as a biological endpoint to examine the possibility of developing skin cancer, as well as the efficacy of sunscreen in preventing such cancer. These studies will be discussed later in the wiki. Since the production of the p53 protein occurs in advance of actual tumor development in UV induced skin cancer, we can look at how inhibiting mutations in the gene from UV radiation can affect subsequent tumor development and skin cancer.

<span style="font-family: 'Times New Roman',Times,serif;"> One of the mutations in the p53 gene is the C-->T and CC-->TT mutation, which are both signature mutations from UV exposure. The CC-->TT occurs when CC is dimerized by UV radiation, which if then replicated, leads to a TT mutation. The [|article], "p53 Tumor Suppressor Gene," discussed the efficacy of sunscreen in inhibiting p53 mutations induced by UV exposure [See Jacey's blog]. The SPF of sunscreen is determined by its ability to prevent sunburn, but is not indicative of how well you are protected from other damages caused by UV exposure. Because p53 mutations correlate with tumor development, researchers looked at sunscreen’s ability to inhibit these mutations from occurring. In one of the experiments, mice were exposed with UVB from solar simulator radiators 5 times per week for 12 weeks. One group of mice received UVB irradiation and sunscreen vehicle control, while another had UVB absorbing sunscreen applied 30 minutes prior to exposure, and the last group had UVB+UVA absorbing sunscreen applied 30 minutes prior. The results showed that mice applied with either sunscreen had an 80%-100% reduction in mutation frequencies for the C-->T and CC-->TT sites in the p53 gene, although the exact numbers are not specified.

=<span style="font-family: 'Times New Roman',Times,serif;">Research: UV-Irradiation and p53 Mutations =

<span style="font-family: 'Times New Roman',Times,serif;">How does sunscreen absorb UV radiation?
<span style="font-family: 'Times New Roman',Times,serif;"> We explored the research done as the authors developed new ways to go about determining that p53 mutations can serve as early biological end-points of photoprotection against an initiating event in UV carcinogenesis. Two years after their initial findings were published, the authors released the results of a secondary experiment conducted using a solar simulator in place of the previously used sunlamp. According to the authors, the sunlamp used in their first experiment had a different UV spectrum than is present in natural sunlight and the new solar simulator alleviated this issue. In addition to the difference in UV emission, the authors note that they did not test if reducing p53 mutations would protect mice against skin cancer development.

<span style="font-family: 'Times New Roman',Times,serif;"> The follow up experiment investigated three interrelated issues: a) Whether p53 mutations come about in mouse skin after exposure to solar simulated UV radiation as opposed to the previously used sunlamp light; b) If there is a possibility of reducing any potentially negative effects by solar simulation on the p53 gene; c) If there is an association between the reduction of UV-induced p53 mutations by sunscreens and protection against skin cancer development using mice as models.

<span style="font-family: 'Times New Roman',Times,serif;"> The authors split the C3H mice into groups based on experimental treatment: UV only, P530 (Vehicle + UV), P531 (UVB-absorbing sunscreen + UV), P532 (UVB-absorbing sunscreen + UV), P533 (UVB- and UVA-absorbing sunscreen + UV), and P534 (UVB- and UVA-absorbing sunscreen + UV).

<span style="font-family: 'Times New Roman',Times,serif;"> **Figure 2.** UV absorption spectra of sunscreen formulations. Monochromatic protection factor spectra of the four sunscreens (P531-P534) and vehicle (P530) obtained by spectrophotometric measurements.

<span style="font-family: 'Times New Roman',Times,serif;">How do we know UV can induce p53 mutations?
<span style="font-family: 'Times New Roman',Times,serif;"> In the article, “Sunlight and skin cancer: Inhibition of p53 mutations in UV-irradiated mouse skin by sunscreens,” researchers studied UV-induced CC-->TT mutations in the p53 gene at codons 154-155 and 175-176 in mice. The CC-->TT mutation is present in 27% of UV-induced skin tumors in mice, and although the C-->T mutation makes up about 70% of p53 mutations, they were unable to identify these single point mutations. Using PCR, they used primers that specifically bound to mutant DNA strands, as well as ones that bound to both mutant and wild-type. Then, by using these amplified PCR product sizes as comparisons, researchers were able to run tests on the mice DNA throughout the trial to determine whether mutations were present or not.

<span style="font-family: 'Times New Roman',Times,serif;"> **Figure 3.** “Amplification of DNA from UV-induced mouse skin tumors with p53 mutations at codons 154-155 or 175-176 by PCR.” //a// depicts the strategy in which researchers used primers to amplify the specific sequences. When the mutant specific primers were used on wild-type DNA, no product formed. 5A and 5B bound to both mutant and wild-type DNA, and produced a 204 bp product, while the mutant-specific primer only bound to the mutant DNA, and produced a 119 bp product. //b// and //c// depict gel separation of the PCR products. Lane 1: 5A/5B primer on tumor DNA, Lane 2: 5A/5B primer on normal DNA, Lane 3b: mutation specific primer at 154-155 of tumor DNA, Lane 3c: mutation specific primer at 175-176 of tumor DNA, lane 4: mutation specific primers on normal DNA.

<span style="font-family: 'Times New Roman',Times,serif;">How can sunscreen inhibit p53 mutations?
<span style="font-family: 'Times New Roman',Times,serif;"> After first developing a way to detect CC-->TT mutations, the article “Sunlight and skin cancer: Inhibition of p53 mutations in UV-irradiated mouse skin by sunscreens,” then looks into how sunscreen can inhibit these mutations. There were 3 different treatment categories of mice used: 1. Vehicle treatment + UVB radiation, 2. SPF 15 UVB absorbent sunscreen + UVB radiation, 3. SPF 15 UVA+UVB absorbent sunscreen + UVB radiation. After 12 weeks, the DNA of the mice were analyzed for the CC-->TT specific mutations at codons 154-155 and 175-176.

<span style="font-family: 'Times New Roman',Times,serif;"> **Table 1.** Number of mice with mutations at each specific codon region for the different treatment groups.

<span style="font-family: 'Times New Roman',Times,serif;"> In their earlier experiments detecting p53 mutations, they found a mutation at codon 148 too, and so this trial includes detection of a CC-->TT mutation at this region. According to the table, it appears that there is a significant decrease in number of mutations between using vehicle treatment and sunscreen. The p value supports the significance in the difference. But the difference between using UVB sunscreen and UVB + UVA sunscreen is not significant. This suggests that UVA does not play a large role in mutations, which could suggest that UVA rays are less harmful than UVB. But also, the experiment was only testing for UVB specific mutations, as well as using UVB radiation, which could skew the data.

<span style="font-family: 'Times New Roman',Times,serif;"> <span style="font-family: 'Times New Roman',Times,serif; line-height: 1.5;">Researchers reasoned that because UVA mutations aren’t as common for UV-induced skin cancers, <span style="font-family: 'Times New Roman',Times,serif; line-height: 1.5;"> but then I wonder if it was necessary to include UVA absorbent sunscreen at all. It’s clear that sunscreen reduced number of mutations, but the way in which it does this is unclear. For example, are there less mutations because sunscreen prevents mutations from occurring or because they attack and get rid of mutants? Researchers suggest that the ability to inhibit p53 mutations is due to sunscreen’s ability to prevent DNA damage, and therefore prevent the mutations from occurring at all.

<span style="font-family: 'Times New Roman',Times,serif;"> **Table 2.** Inhibition of UVB-induced p53 mutations in C3H mouse skin by sunscreens.

<span style="font-family: 'Times New Roman',Times,serif;"> This table lays out the results done by the research conducted two years later by the same authors. The numbers serve as proof that the sunscreens used in this study inhibited the number of p53 mutations induced by UVB by 80%-100%, a quantifiable measurement that can be used to determine the effectiveness of certain sunscreens.

**<span style="font-family: 'Times New Roman',Times,serif;">How can p53 mutations be an early indicator of skin cancer? **
<span style="font-family: 'Times New Roman',Times,serif;"> In “Sunlight and skin cancer: Inhibition of p53 mutations in UV-irradiated mouse skin by sunscreens,” researchers analyzed p53 mutations in mice to determine the earliest time point these mutations could be detected, and their relation to later development of tumors.

<span style="font-family: 'Times New Roman',Times,serif;"> **Table 3.** Number of mice with mutations at each specific codon region. <span style="font-family: 'Times New Roman',Times,serif;"> The earliest mutation was detected at week 4, which was when the first analysis was run. Researchers determined that mice with tumors of 10x10mm in size would be killed, which is most likely why there is one less mouse at week 16. Because there was a mutation at week 4, which was their first test, it is possible that the mutation could have occurred even earlier. So for future studies, it would be helpful to start tests more in advance, maybe even from week 1 just to ensure you detect the mutation at the earliest time point. The fact that non of the non-irradiated mouse skin samples had a p53 mutation also supports the idea that UV induces p53 mutations.

<span style="font-family: 'Times New Roman',Times,serif;"> **Figure 4.** Gel separation of PCR products obtained after 12 weeks in determining earliest detection of p53 mutations. a depicts products formed by mutant specific primers for codons 154-155, b depicts products formed by mutant specific primers for codons 175-176. Unirradiated mice DNA (lanes 1 &2), and UVB irradiated mice (lanes 3-12), are depicted in a and b. 154-155 mutants (c) and 175-176 mutants (e) were reamplified and resequenced, d and f are their wildtype DNA strands. Arrows point to bands not found in unirradiated mice.

<span style="font-family: 'Times New Roman',Times,serif;"> By looking at the gel electrophoresis of the PCR products found when analyzing p53 mutations of the mice, there appears to be 2 mutants at codons 154-155 and 3 at codons 175-176, which correlates with the information found in Table 1. When comparing the gel in figure 3a with figure 1b, there should be a band of 119 bp. When comparing figure 3b with figure 1c, there should be a band of 55bp. Both figure 3a and 3b have bands near the bottom, which could be the corresponding bands to figure 1, but they also have bands near the top. When the PCR products for these mutants were reamplified, we see that there was a CC-->TT mutation for both codons, as indicated on the image. When comparing the other bands on the mutant products with their wild-type image, it appears that the only mutation present is the CC-->TT.

<span style="font-family: 'Times New Roman',Times,serif;"> **Figure 5.** Percent of mice affected by p53 mutations (circles) and skin cancer tumor development (triangles). Each plot for mice affected by p53 mutations comes from 10 mice, while data for tumor development is based on 30 mice.

<span style="font-family: 'Times New Roman',Times,serif;"> Once researchers were able to confirm the CC-->TT mutated codons in mice, they could then plot incidence of mutations along with later detection of tumors. Figure 2 shows that after about 30 weeks, all UV-irradiated mice developed tumors, but only about 50% of the mice had a CC-->TT p53 mutation at the end of the trial. But because they were only looking at one specific mutation, the frequency was most likely higher if you take into account other mutations that could have also been occurring. But it is difficult to accurately say whether there were other mutations, and specifically which mutations were also in the mice. The first tumor was detected at about week 16, whereas the first mutation was detected at week 4. Plus, based on the trend of both lines, it appears that the mutations occur before the onset of tumors, and so we agree with the researchers’ claim that p53 mutations can be used as early indicators for tumor development.

<span style="font-family: 'Times New Roman',Times,serif;"> **Figure 6.** "Detection of p53 mutations at codons 154-155 in UVB-irradiated mouse skin by allele-specific polymerase chain reaction and SCCP. DNA was isolated from mouse skin, amplified using mutant-specific primers and an aliquot of the PCR product was subjected to SCCP analysis."

<span style="font-family: 'Times New Roman',Times,serif;"> According to the authors, the DNA from the UV-induced mouse skin tumors are known to contain the specific 154-155 codon mutations. Gene amplification tests were used, where the amplified regions indicate mutations in the p53 gene. Primers specific for mutant p53 sequences at codons 154-155 amplified the DNA from the skin of 3/8 UV-irradiated mice, 3/8 vehicle/UV-irradiated mice, and did not amplify the DNA from unirradiated mouse skin. The DNA from tumors known to have mutations at codons 154-155 resulted in highly amplified bands (lanes distinguished by +). So what does this tell us? This data shows that the sunscreens used in this study inhibited the number of UVB-induced p53 mutations and that there were no incidences of p53 mutations in the mouse skin that was not exposed to UV radiation.

<span style="font-family: 'Times New Roman',Times,serif;"> **Figure 7**. "Sunscreen application prevents UV-induced skin cancer induction. Sunscreens were applied to the dorsal skin of mice 30 minutes before each UV exposure."

<span style="font-family: 'Times New Roman',Times,serif;"> The probability of tumor development was calculated for groups of mice exposed to: UV only (squares); vehicle plus UV (diamonds); sunscreen P531 plus UV (open circles); sunscreen P532 plus UV (open triangles); sunscreen P533 plus UV (open squares); sunscreen P534 plus UV (open diamonds); and plotted versus the cumulative dose of solar simulated UVB radiation received by the mice. With the topical application of sunscreen formulas to the skin, the probability of tumor formation does not exceed 0.25. These findings reinforce the idea that sunscreens are able to contribute to skin cancer prevention. It can be implied from this data that p53 mutations have the ability to serve as more quantifiable end points that measure promotional events in the multistep process of carcinogenesis.

=<span style="font-family: 'Times New Roman',Times,serif;">Summary and Further Explorations =

<span style="font-family: 'Times New Roman',Times,serif;">Based on these two articles, it is clear that p53 mutations can be induced by UV radiation since there were no mutations for non-irradiated mice, and multiple mutations for radiated mice. Moreover, the researchers were able to support their claim that mutations occur before tumor development, although the way in which detecting these mutations can be used as an early indicator of skin cancer is still questionable since the earliest time point of detection was not identified. Another thing to take into consideration is that researchers only looked at one mutation in the p53 gene; the CC-->TT at codons 148, 154-155, and 175-176. They reasoned that this mutation makes up for about 27% of all p53 mutations in C3H mice, which raises the question of if this is the same for humans, and how they can then relate their research findings on mice to human skin. But because these articles are from over 10 years ago, at the time they might not have had more information on other genes to study.

<span style="font-family: 'Times New Roman',Times,serif;"> Another thing researchers left unclear was whether all mice who developed a mutation later developed tumors. According to figure 3, it appears as though all mice had tumor development after week 30, they only had 10 mice for noting p53 mutations, while they had 30 mice in determining tumor development, which raises the question of whether the mice they observed for tumor development were the same they observed for mutations. If so, why weren't the other 20 mice included into the p53 mutation plot?

<span style="font-family: 'Times New Roman',Times,serif;"> When analyzing the experiments done to test the effectiveness of sunscreen, their data shows a significant difference between mice applied with sunscreen, and mice applied with only the vehicle treatment. One thing that did not seem to make a difference was whether the sunscreen had UVA absorbers in it, and it's also debatable why they used this in the first place since they were using UVB radiation.

<span style="font-family: 'Times New Roman',Times,serif;"> Overall, sunscreen definitely made a difference in whether p53 mutations occurred, but what is left uncertain is how it does this, as well as the frequency in which the mice who developed mutations also developed tumors over time. The article suggests that sunscreen can consequently prevent photocarcinogenesis, but none of the data shows a direct correlation, although the reduction of p53 mutations does suggest that this could be a possibility. In future studies, researchers could look into analyzing the entire gene for mutations, or studying other genes besides p53, such as those listed in the image below.

<span style="font-family: 'Times New Roman',Times,serif;"> **Figure 8.** Frequency of DNA mutations in melanoma. Source: <span style="font-family: 'Times New Roman',Times,serif;"> http://cancergenome.broadinstitute.org/index.php?ttype=MEL

<span style="font-family: 'Times New Roman',Times,serif;"> Based on the conclusions above, we believe that we have strong evidence supporting the idea that the detection of p53 mutations is a better indicator of sunscreen effectiveness than the presence of erythema. Mutations in the p53 gene, specifically the dimerization of the CC pairs in the DNA sequence, are necessary steps on the pathway towards the development of cancer. p53 mutations are a more direct molecular cause for cancer, while erythema is part of a series of indirect steps that have a weaker correlation with skin cancer. Sunburns are complex physiological responses to DNA damage due to UV radiation. They are measured by a subjective determination, "redness", whereas the mutation in the p53 gene is quantifiable. We see it as more logical to measure sunscreens based on how well the formulas protect us from p53 mutations rather than some difficult to understand physiological response. The presence of a p53 mutation can be used to deduce the effectiveness of sunscreen, information which provides a foundation for researchers to determine more efficacious modes of skin cancer prevention.

<span style="font-family: 'Times New Roman',Times,serif;"> We suggest developing more biologic end points to measure initiating events in the multistep process towards skin cancer. With these additional endpoints, researchers may then be more able to fully assess the role of sunscreens in protecting against skin cancer development.

=<span style="font-family: 'Times New Roman',Times,serif;">References =