"Read More About It" Pages - Biology 376 - Fall 2009

Dec. 9, 2009 Presentations

The Inherent Instability of Mutant p53 is Alleviated by Mdm2 or p16INK4a Loss

Presented by Mitchell Mendez

Any mutation in the p53 gene can lead to unregulated cell growth potentially resulting in cancer. The most common mutation found in the p53 gene is in the form of a missense mutation in the DNA-binding domain. In addition to mutations, the function of p53 can also be altered by proteins such as Mdm2 and p16INK4a. A negative regulator of p53 is the gene, Mdm2, which encodes a nuclear phosphoprotein of the same name that binds and inhibits the transactivation of the p53 protein. Overexpression of Mdm2 can result in inactivation of p53 which prevents its normal tumor suppressor function. Mdm2 is also an E3 ubiquitin ligase that targets p53 for degradation. The p16INK4a tumor suppressor protein, encoded by the CDKN2A gene, is an inhibitor of Cyclin-D/CDK complexes that phosphorylates Rb. Inhibiting the phosphorylation of Rb induces cell cycle arrest. A mutation in the p16INK4a protein increases the risk of developing cancers including melanoma which may develop as a result of an inactivating mutation in the p16 gene.

Terzian et al. produced mice that had a missense mutation at codon 172 which is homologous to the human hot spot mutation at amino acid 175 which is known to promote cancer. Mice that were homozygous for the mutant p53 gene had unstable mutant p53 in normal cells and stable mutant p53 in only some tumor cells. Mutant p53 mice were found to have low levels of the mutant p53 protein in normal tissues and some tumors unless Mdm2 or p16INK4a were absent. These mice, which had low levels of p53 in their normal tissues, exhibited high levels of p53 in 79% of their tumors. Because of the low levels of mutant p53 in both normal tissues and some tumors, it was hypothesized that either levels of mutant p53 in both normal tissues and some tumors, it was hypothesized that either Mdm2 was present in sufficient amounts to block the function of p53 or that there was some unidentified reason that caused the degradation of p53 in normal cells and some tumors.

It was discovered that homozygous mutant mice that lacked Mdm2 exhibited increased mutant p53 levels, increased occurrence of tumors, decreased survival, and a metastatic phenotype in comparison to mice that were homozygous for only the mutant p53 and with p53/Mdm2 double-null mice. The loss of p16INK4a was shown to have a similar effect to that of Mdm2 as it stabilized mutant p53 in vivo and yielded a metastatic phenotype. Mice that lacked Mdm2 or p16INK4a stabilized mutant p53 and developed tumors at an earlier age than the p53 mutant mice while also demonstrating a gain-of-function metastatic phenotype. To determine whether DNA damage would also increase the levels of mutant p53 protein, Terzian et al. exposed mice to whole-body irradiation and then examined cells from their spleens and thymuses. They discovered that radiation also had a stabilizing effect on mutant p53.

In conclusion, it was found that mutant p53 is regulated by Mdm2 or p16INK4a loss in a similar manner as wild-type p53 and its stabilization contributes to its gain-of-function phenotype. Terzian et al. also demonstrated that the mutant p53 gene was intrinsically unstable in normal cells and that tumor-specific changes helped stabilize the mutant p53. Any drug that affects the interaction between Mdm2 and wild-type p53 may also come to affect the interaction between Mdm2 and mutant p53 thus negatively affecting tumor metastases in vivo. Eliminating the stability of the mutant p53 gene may also come to suppress the metastatic phenotype.

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Declining p53 Function in the Aging Process: A Possible Mechanism for the Increased Tumor Incidence in Older Populations

Presented by Jessi Nerland

It is a widely accepted hypothesis that cancer is seen more commonly in older individuals because individual cells accumulate mutations over a lifetime. However, this paper proposed an alternative hypothesis: p53 function declines with age which leads to a greater frequency of mutations and tumorigenesis in the aging process. P53 responds to several stress signals in the cell and has a number of different responses including transcriptional regulation of target genes, apoptosis, and cell cycle arrest. A disruption of p53 can lead to tumor development or progression.

The first aspect of p53 activity that the paper explored was p53 transcriptional activity in aging mice. C57BL/6 mice of different ages were irradiated with ë-radiation to induce p53 activity. P53 target genes mRNA levels were measured before and after irradiation. The induction folds of genes were consistently lower in aging mice. For females, the onset of aging is at 20 months and for males at 28 months. These results not tissue specific. The decline in transcriptional activity was seen in other mouse strains including DBA2 and BALB/c as well. The second aspect of p53 activity that the paper looked at was p53-dependent apoptosis in aging mice. They found in the C57BL/6 strain of mice that there were significantly fewer apoptotic cells in 28 month males and 20 or 25 month females than 6 month old mice. These results were also found in the DBA2 and BALB/c strains. The paper then looked at a decreased p53 protein accumulation in aging mice. They found that p53 protein levels were induced at a greater level in young mice than older mice in the C57BL/6, DBA2, and BALB/c strains. The protein levels decreased in 25 month old females and 28 month old males. Since the mRNA levels of p53 negative regulators did not change, results indicated that negative regulators were not at fault for the decline of p53 function in aging mice.

ATM kinase activates p53 function and when there is a deficiency in ATM function there is a deficiency in p53 function. ATM protein levels and activity decreased in older mice for C57BL/6, DBA2, and BALB/c strains. These findings suggest that the p53 response to irradiation declines with age due to the declining ATM function with age. Further results showed that p53 activity did not just decline because there was a general decline of all signal transduction pathways with age. Other signal pathways, such as IGF-1-AKT and mTOR, maintained the same activity throughout the aging process. These pathways aid in regulation of longevity and tumorigenesis. The researchers then focused on whether p53 function would decline in aging mice in response to stress signals other than irradiation. The stress signals they used included etoposide and adriamycin which both induce double-stranded DNA breaks like irradiation, low levels of actinomycin which blocks RNA synthesis, ALLN which inhibits degradation by the 26 S proteasome and Taxol which disrupts microtubules. The amount of apoptotic cells and levels of p53 protein accumulation were lower in older mice than younger mice for all stress signals. However, the amount of difference changed for different stress signals. There was a greater difference in irradiation, etoposide, adriamycin, and ALLN and a lesser difference in actinomycin D and Taxol.

This paper concluded that the increase in tumors in older populations might be due to declining p53 function. The decline in the p53 function may be due to the decline in ATM activity rather than a general decline in all signaling pathways.

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Presented by Rachel Oyama

It has been observed over the course of cancer research that different types of cancers are prevalent at different ages. Although this is a well documented phenomenon the reason behind it is not well understood. In this paper it is hypothesized that a specific oncogene, in this case MYC, may only be capable of causing tumorigenesis singlehandedly at certain types of development.

MYC (formally called C-MYC) is a proto-oncogene that encodes a transcription factor. This transcription factor is important in the development in tumors and cancers because it controls cell growth and proliferation by regulating a wide variety of genes that control these stages of cell development. MYC over-expression is a common factor in the growth and proliferation of tumors because such over-expression leads to the unchecked development and propagation of cells. Not only does it cause an overabundance of cellular growth, MYC also inhibits the differentiation of these cells.

In this study the scientists wanted to see if MYC over-expression had different consequences with regards to the development of tumors in the liver based on when in development the over-expression occurred. To answer this question transgenic mice were used so that the expression of MYC could be conditionally regulated. These mice were looked at three specific stages, embryonic, neonatal, and adult.

It was found that when MYC was over expressed in both embryonic and neonatal mice, tumor growth occurred relatively quickly. Embryonic mice experienced tumor growth within 10 days of birth while neonatal mice experienced neoplasia within 8 weeks of over expression. In adult mice, the onset of tumorigenesis was much slower with the growth of tumors not presenting until 15-35 weeks after MYC had been overexpressed. In adult mice it was found that MYC caused a great amount of DNA replication, but that because mitosis did not occur, tumors did not grow. This indicates cells of embryonic and neonatal mice are in the stages of growth and proliferation and are more likely to be affected by the over expression of MYC alone. Older mice on the other hand, have cells that are no longer growing or proliferating at a high rate and instead have fail safes and developmental checkpoints that ensure that the over expression of MYC alone does not cause tumorigenesis.

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Romidepsin Inhibits Ras-dependent Growth Transformation of NIH 3T3 Fibroblasts and RIE-1 Epithelial Cells Independently of Ras Signaling Inhibition

Presented by Raneeza Cano

The Ras protein is involved in cellular signal transduction. When bound to GTP, Ras is activated and can signal cells to grow and divide. To be switched off, Ras must bind to GDP. Ras has a low intrinsic abiliy to convert GTP to GDP, however, so it requires GTPase-activating proteins (GAPs) to speed up that conversion. Thus, if Ras has a mutation that prevents GTP from hydrolyzing into GDP, the Ras protein remains active and continues to signal cells to keep dividing and growing. The three Ras oncogenes that are mutationally activated in about 30% of cancers include, HRAS, KRAS, and NRAS.

In this paper, Hanker and colleagues wanted to further examine previous studies which suggested that romidepsin, an inhibitor of tumor growth, could prevent Ras-dependent signaling and growth transformation. To do this, one of their main objectives was to test whether romidepsin has to directly affect the Ras signaling pathway in order to block the growth of **Ras-transformed cells. **A Ras-transformed cell, an ErB2/Neu-transformed cell, and a Raf-transformed cell are all abnormal cells and potentially on their way to being cancerous cell. These Ras-, ErB2/Neu-, and Raf-transformed cells were infected with a retrovirus containing the activated versions of Ras, ErB2/Neu, or Raf, respectively. Being treated with such active versions, the infected NIH 3T3 or RIE-1 cells are continuously growing, which is why they become abnormal or transformed cells.

NIH 3T3 mouse fibroblasts were infected with a retrovirus containing oncogenic forms of either H-, K-, or N-Ras, as well as oncogenic B-Raf or ErB2/Neu. This was then followed by a treatment of romidepsin into those infected NIH 3T3 cells. The results showed that treatment with 3-5nM of romidepsin reduced anchorage-dependent proliferation of all the NIH 3T3 Ras-transformed cells and ErB2/Neu-transformed cells. The growth inhibition of ErB2/Neu-transformed cells is consistent with previous studies (as you may recall, those studies suggested that romidepsin could block Ras-dependent growth transformation) because ErB2/Neu transformation of NIH 3T3 cells requires Ras. On the other hand, B-Raf-transformed cell growth was also found to be inhibited by romidepsin even though Raf cell transformation is indepdent of Ras; These results suggest that although romidepsin can inhibit the proliferation of Ras-transformed cells (Ras-dependent cells), it does not do so by affecting the Ras signaling pathway since romidepsin can also reduce cell growth of Ras-independent cells. Therefore, romidepsin must be inhibiting proliferation of NIH 3T3 cells by ways of another mechanism other than blocking anything in the Ras signaling pathway. In addition to NIH 3T3, RIE-1 epithelial cells were also infected with a retrovirus containing oncogenic forms of either H-, K-, N-Ras, B-Raf or ErB2/Neu and then treated with romidepsin. Like the transformed NIH 3T3 cells, the results showed that romidepsin also reduced the anchorage-dependent proliferation of all the oncogene-transformed RIE-1 cells. On the other hand, unlike NIH 3T3 cells, Hanker and colleagues observed that romidepsin reduced proliferation of even RIE-1-nontransformed cells (the control cells). This suggests that depending on the cell type, there is a lot of variability in response to romidepsin.

In conclusion, the results show us that Ras-transformed cells are affected by romidepsin; these results are supported by other studies. The results also suggest that although romidepsin does block Ras-dependent transformation, it also blocks transformation induced by other oncogenes. For that reason, it is suspected that response to romidepsin will not be limited to tumors that have RAS mutations. Additionally, the striking differences that were observed between NIH 3T3 cells and RIE-1 cells brings to light the importance of using more than one model cell type when studying such experiments - because like romidepsin, some products may not affect different cell types in the exact same way.

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Dec. 2, 2009 Presentations

Increased Wnt Signaling Triggers Oncogenic Conversion of Human Breast Epithelial Cells by a Notch-dependent Mechanism

Presented by Kaitlyn Howell

Breast cancer is known to be caused by a series of gene mutations. The role of mutated genes in the creation of tumors in breast cancer has been hard to determine because research can only start when the tumor is already present. In this paper, transgenic mice with human mammary epithelial cells (HMECs) were used to examine the role of the upregulation of Wnt to create a DNA damage response (DDR) and activate Notch signaling pathway in human breast cancer to create a tumor.

The Wnt signaling pathway begins with a Wnt ligand binding to the receptor, frizzled, to stop the degradation of β-catenin. β-catenin can then move to the nucleus where it can form complexes with transcription factors to activate genes. To create the upregulation of the Wnt signaling pathway, Wnt-1 was ectopically expressed in HMECs. The cells with excess Wnt-1 (Wnt-1-HMECs) were able to continue to divide when the control cells started to die. To test if Wnt-1-HMECs cells were tumorigenic, they were placed into the mammary glands of mice. Within two months, 68% of the mice had tumors.

The upregulation of Wnt signaling pathway creates a DDR through increased phosphorylation of two proteins, Chk2 and H2AX. DDR inactivates the G1/S, p-16/Rb and p53 checkpoints. The cell can move from the G1 phase to the S phase of the cell cycle after pRb is phosphorylated by a combination of different cyclins. The Rb mechanism was found to be disrupted by increased cyclin D3, cyclin A, cyclin B1and p-16 levels using immunoblotting. The stabilization of p53 is also a consequence of DDR to increase the concentration of p53, a known oncogene.

Although DDR causes instability, the transformation of RBP-Jk/CBF-1 from a transcriptional repressor to an activator in the Notch signaling pathway is necessary for tumorigenesis. The Notch signaling pathway begins with a membrane-bound ligand binds to a notch protein on an adjacent cell which causes two cleavages of the receptor to release the Notch intracellular domain. The Notch intracellular domain then moves to the nucleus where it binds to a transcription factor to form a transcriptional activation complex. Although the Notch signaling pathway is necessary for tumorigenesis, it must work in conjunction with the Wnt signaling pathway.

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Antisense Gapmers Selectively Suppress Individual Oncogenic p73 Splice Isoforms and Inhibit Tumor Growth in vivo

Presented by Kari Milam

Due to structural and functional similarities, human p73 has been identified as a member of the p53 tumor suppressor family although p73 is usually not mutated in human cancers. The gene TP73 normally encodes for the full-length, tumor suppressing TAp73 protein but can produce multiple oncogenic NH2-truncated transcripts (also called p73 variants, isoforms, or splice forms) through differential mRNA splicing and alternative promoter usage. Collectively referred to as DNp73, these p73 variants lack the transactivation domain responsible for the tumor suppressing function of the full-length TAp73 protein and therefore inactivate major tumor suppressor pathways in human cells. In various human tumor cells these abnormal NH2-terminally truncated p73 variants are frequently elevated yet are not found in surrounding, normal tissues. Advanced tumor stages correlate with overexpression of particular p73 variants.

Although it is evident that individual p73 splice variants play a crucial role in tumorigenesis, no attempt has yet been made to selectively silence these isoforms without also knocking out the normal TAp73 apoptotic full-length form. The goal of this research was to develop specific silencers for these NH2-truncated, alternatively spliced p73 isoforms in order to knockdown oncoproteins in growing human tumors. In these experiments, RNAi technology is used to create anitsense oligonucleotides designed specifically against each amino-terminal alternatively spliced p73 isoform.

Locked nucleic acid (LNA) antisense oligonucleotide (ASO) gapmers were created complementary to the exon splice junctions of each variant p73 isoform. Each ASO is composed of four LNA monomers flanked by a 12 nucleotide region of DNA directed against a variant transcript. The success of these ASOs was experimentally determined through assessing their knockdown effect of mRNA levels in tumor cells, their induction of apoptosis, and their activity in on tumors in vivo. Cancer cells treated with specifically designed ASOs showed a strong and specific reduction in tumorigenic p73 transcripts and also oncogenic p73 proteins without eradicating the wild-type tumor suppressor full-length form of p73. Melanoma growth was also shown to be significantly inhibited by ASO-116 coupled to a magnetic nanobead carrier.

The strong correlation between abnormal and overexpressed p73 transcripts red flagged them as markers for disease severity and targets for cancer intervention. The LNA-ASOs (antisense gapmers) devised in this experiment were shown successful in selectively differentiating between the p73 oncoprotein variants. This study provides new tools with which to discern the biological characteristics of these abnormal and overexpressed p73 isoforms in various human cancers. These LNA-ASOs may be useful in therapeutic cancer targeting, as well.

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p19(Arf) Suppresses Growth, Progression, and Metastasis of Hras-Driven Carcinomas Through p53-Dependent and -Independent Pathways

Presented by Erin Davis

The expression of an oncogene, such as Ras, has been shown to induce a pathway of tumor suppression by activating the tumor suppressor p19Arf, which in turn induces the expression of p53 and results in the inhibition of further tumor development. The purpose of this study was to examine the functional interactions between Ras, and the two tumor suppressors p19Arf, and p53.

Cancer-causing mutations on the Ras gene result in a constitutively active Ras protein, wreaking havoc on the pathways that regulate cellular proliferation, apoptosis, differentiation, senescence, adhesion, and migration. p53, one of the most frequently mutated tumor suppressor genes in human cancer, is activated in response to cellular stresses including oncogene activation. Once activated, stabilization modifications occur that promote p53’s ability to act as a transcription factor, but when mutations are present and the function of p53 is lost, impaired apoptosis and tumor progression occur. Recently, p19Arf has been established as a connection between Ras mutation activation, and p53 tumor suppression. Like p53, p19Arf is also induced by oncogene expression. In cells deficient in p19Arf there was also reduced activation of p53, allowing for a more efficient instance of tumorgenesis, benign to malignant transition of the tumors, and metastasis.

Experiments using mice treated with a two stage tumor-inducing chemical treatment called DMBA/TPA were performed to establish the p53-dependent and -independent pathways in which p19Arf contributed to the suppression of tumorgenesis, tumor growth, malignant progression, and metastasis. Results show that in early stages of tumor development, loss of one or both of the p19Arf alleles leads to accelerated tumor growth, and also exhibits a gene dosage effect. It was shown that at this stage, p19Arf does not regulate p53. The role p19Arf plays in benign to malignant transition and metastasis is, however, shown to be p53-dependent. Also, Favorable p53 mutations for tumor progression are directly related to the initial activating mutation in Ras, as mediated by p19Arf through a p19Arf / p53 signaling pathway.

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Nov. 23, 2009 Presentations

Inhibition of Cyclin-Dependent Kinases is Neuroprotective in 1-Methyl-4-Phenylpyridinium-Induced Apoptosis in Neurons

Presented by Chardonnay Shinn

Mitochondrial dysfunction has been linked to many neurodegenerative diseases including Parkinson’s disease. The mitochondrial complex I respiratory chain is the largest enzyme of the oxidative phosphorylation system. When this pathway’s function is altered or inhibited, there can be a large influx of reactive oxygen species (ROS). Mitochondria generally use oxygen to create energy in the form of ATP, releasing some of this oxygen in the process. However, when too much is released, these species can become unstable and react with anything in the cell. In this study, researchers tested the effects of the neurotoxin 1-methyl-4-phenylpyridinium (MPP+) on complex I function, which was shown to induce apoptosis via neuronal reentry into the cell cycle.

Experimenters observed an increase in cell cycle proteins after MPP+ induction, including cyclin D, cyclinE, cdk2, cdk4, and the transcription factor E2F-1, using Western-blot and immunocytochemistry analysis. In addition, researchers measured caspase activation in MPP+, specifically caspase-3, via a colorimetric substance, which was a synthetic substrate of caspase-3. Levels of apoptotic nuclei were measure by propidium iodide staining.

Researchers determined that the influx in reactive oxygen species, measured by a fluorescent probe, was the major result of complex I inhibition/alteration, causing neurons to reenter the cell cycle. Results of the Western-blot showed that transcription factor E2F-1, which is a know pro-apoptotic transcription factor, was the likely cause of apoptosis following neuronal reentry into the cell cycle, with high concentrations in nuclear and cytoplasmic locations. Though other studies have shown that caspase activation is largely involved in the apoptotic process, this study failed to show that the amount of activation of caspase 3 by MPP+ was significant enough to induce apoptosis. In addition, the tested caspase inhibitor zVAD-fmk did not reduced apoptosis. Therefore, they concluded that there were other caspase-independent pathways that were responsible for the apoptotic process.

These experimenters found that MPP+ induces apoptosis via inhibition of complex I, which increases amounts of ROS and cell cycle proteins, specifically TF E2F-1. Finally, they demonstrated that certain drugs/vitamins, specifically Flavopiridol, an inhibitor of cyclin-dependent kinases and vitamin E, an antioxidant, can be used to diminish MPP+ induced apoptosis, and possibly neuronal apoptosis in Parkinson’s disease patients in general.

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Decreased Expression of Pro-apoptotic Bcl-2 Family Members during Retinal Development and Differential Sensitivity to Cell Death

Presented by Maesa Hanhan

Neurodegenerative disorders, including Retinitis Pigmentosa (RP), involve the degeneration of neural development by use of control points in photoreceptor apoptosis. Retinal cells are produced in excess during development. Therefore, they are eliminated by apoptosis when they fail to make the appropriate connections, such as photoreceptor cones, that are crucial for normal development. When the retina matures, mechanisms must be mediated in order to avert inappropriate cell death.

In this study, the retina of pre- and post-natal mice were analyzed to determine the involvement of pro-apoptotic Bcl-2 family members in photoreceptor degeneration using light-induced damage to the retina. Also, mitochondria were used to observe whether they do provide a control point in mediating cell death by releasing pro-apoptotic factors, particularly cytochrome c, which activates caspase-9. The activation of caspase-9 was assessed at different times after exposure to light, using TUNEL, to determine if dying cells would employ this mitochondrial cell death pathway. The caspase-9 was present for all different times after exposure, indicating that this protein is used to implement the mitochondrial pathway of apoptosis.

In addition to these pro-apoptotic proteins, others including AIF, Omi/HtrA2, and endoG reside in the mitochondrion and are capable of providing a capase-independent pathway. To explain what factors were executing apoptosis in the absence of mitochondrial factors, the levels of pro-apoptotic and anti-apoptotic factors were studied. Pro-apoptotic Bcl-2 proteins were found at low levels in adult photoreceptors and did not increase during light exposure. Therefore, it determined that a major difference existed between the levels of these two apoptotic factors in immature and mature retina.

To test the hypothesis that the sensitivity of the retina to the mitochondrial pathway of apoptosis decreases with age, death stimuli that target mitochondrion were applied to retinal explants at different stages of maturation. Results show that TUNEL positive cells were abundant in early post-natal explants, but the adult retinal explants had reduced expression of the pro-apoptotic proteins. Therefore, it can be concluded that adult photoreceptors are more resistant to apoptosis than the pre-natal or early post-natal photoreceptors and that these cells can execute caspase-independent apoptotic pathways, while caspases are used to implement death in early development.

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ATM Promotes Apoptosis and Suppresses Tumorigenesis in Response to Myc

Presented by Stephanie Papp

A mutation in the gene Myc causes many different types of cancer, making it an oncogene. The normal function of this gene is as a transcription factor that helps to regulate the cell cycle, and when mutated, the gene can cause mitotic failures in regulation leading to cancer, as well as damage to DNA strands, which can also result in cancer. Mutations in the gene normally result in excess expression, which has been found in multiple tumor types. Concurrently, the phosphorolation of a second gene, ataxia-telangiectasia mutated kinase, or ATM, has been detected in the earliest developing cancers. ATM operates in the cell cycle by phosphorolating a very well-known tumor suppressing gene, p53 along with other genes, such as Mdm2, which can activate P53, as well. This paper investigates a potential relationship between the Myc gene and the ATM gene that could be very significant in approaches to fight cancer if ways to increase regulation of these genes can be found and their specific roles in the prevalence of cancer could be understood.

In this paper, Pusapati and his colleagues use K5 Myc transgenic mice that have over-expressed Myc to show that when Myc is in excess in mice, increased p53 phosphorolation is the result. They show this by comparing the transgenic mice with normal, untreated mice, and irradiated mice, radiation being a treatment that would lead to tumor growth. The results clearly showed an increased presence of phosphorolated p53 in the transgenic and IR mice, but not in the untreated mice. Thus, the scientists concluded that increased Myc had a similar cancer causing property to radiation, which activated the p53 gene. To find the role of ATM in this pathway, the researchers used cells from normal tumors as well as cells from tumors from individuals who carried the ataxia-telangiectasia, or AT, disease, in which the ATM gene is missing or mutated, and irradiated them. The normal cells showed evidence of phosphorolated p53 while the AT cells did not, providing evidence that ATM was a necessary intermediate to activate the p53 in response to excess Myc. In addition, the authors furthered their study by showing that Myc activates the ATM response through its destruction of DNA strands using experiments in which ATM markers appear at the break sites as evidence of ATM activity, and that Myc is in fact what damages the DNA by comparing the transgenic and normal mice to see which exhibited more DNA damage. Lastly, they showed that an absence of the ATM gene might actually promote tumor growth in both epithelial and lymphoma cancers, in experiments with ATM negative mice that exhibited faster tumorigenesis than mice who carried the ATM gene.

All of their experiments led to the conclusion that an increase in Myc expression damages DNA strands, activating the ATM gene, which then in turn phosphorolates the p53 gene, causing apoptosis.

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PKR, a p53 Target Gene, Plays a Crucial Role in the Tumor-suppressor Function of p53

Presented by Rusty Wall

This paper was written to determine PKR’s role in the tumor suppressor function of p53. P 53 is the most common cancerous mutation found and studied as we have learned in class. It is probably the most common cancerous mutation because it usually controls development through the cell cycle. P53 is notorious because it helps repair DNA by stopping and stalling the cell process in the G1 checkpoint. This stall is imperative because it allows the cell to repair itself. If the cell does not repair itself, p53 promotes the cell to undergo apoptosis, which is another incredibly significant cell process to a successful life. Regulation of such a process defines cancerous or hazardous cells. The altercations in the p53 could potentially kill an organism due to synthesis of damaged, unhealthy DNA and/or failure to terminate the harmful DNA. Clearly p53 is an important protein. If PKR influences the p53 protein or its normal functions in any way, it is also highly significant in cancerous research, which is why we must study this target gene and its expression.

Cheol Yoon and his research team found that PKR plays a significant role in the tumor suppressor function of p53 during the activation of intracellular networks. They came to this conclusion after a series of experiments and analysis. They used immunocytochemistry, Luciferase assays, electrophoretic mobility shift assay, western blot analysis, Real-Time Quantitative RT-PCR Analysis and Chromatin Immunoprecipitation Assay to support and determine their opinions on PKR. At first they examined how p53 influences PKR and whether or not it acts directly on the PKR promoter. They also tested to see if PKR can be induced independently of type 1 IFN, a protein that triggers the immune system in response to pathogens. Then the scientists performed experiments on PKR’s affects on the roles of the p53 protein and its normal functions such as cell apoptosis and inhibition of translation to damaged DNA. They also performed test on animals to witness the effects PKR had on human colon cancer cells. In the end, they were able to construct the map displaying PKR’s location, pathway and what it influences.

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Nov. 18, 2009 Presentations

L1 Cell Adhesion Molecule is Neuroprotective of Alcohol Induced Cell Death

Presented by Rachelrose Heinzel

The L1 protein is a member of the Immunoglobulin Superfamily Adhesion protein family (Ig superfamily). The L1 specific molecule is found in the central nervous system which plays a key role in the developmental process of a human or mouse embryo's central nervous system. The L1 protein performs many different functions such as cell adhesion, neurite growth, neural migration, and axon fascicle formation. In the paper the authors, Rose Gubitosi-Klug, Corena G. Larimer, and Cynthia F. Bearer, correlated L1 cell mutations and FAS where they have similar phenotypic abnormalities as seen in humans and experimented in rats and mice.

Rose Gubitosi-Klug, Corena G. Larimer, and Cynthia F. Bearer performed their experiment on rat cerebellar granule cells that were taken from six rat pups and were prepared for culture. Next, the L1-Fc was prepared in order to create an extracellular domain of L1 and control slides were also made to set the standard. Once the cultures are prepared and the experiment begins, ethanol is added to the cultures which are observed and data is then measured for cell death and neurite outgrowth at 48, 72, and 120 hours.

Rose Gubitosi-Klug, Corena G. Larimer, and Cynthia F. Bearer determined that ethanol decreases the amount of cell survival at the 48 hour period and it reduced the amount of cell survival when L1-Fc was on the culture. In conclusion, Rose Gubitosi-Klug, Corena G. Larimer, and Cynthia F. Bearer discovered that L1 protein does in fact cause cells to live when ethanol was introduced and, finally,as mentioned earlier, L1-Fc actually causes the cell survival rate to decrease when ethanol is present.

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The Nerve Growth Factor Precursor ProNGF Exhibits Neurotrophic Activity But is Less Active Than Mature Nerve Growth Factor

Presented by Jennifer Rosin

Neurotrophins are a class of proteins that stimulate neurons to divide, grow, or differentiate. Nerve growth factor (NGF) is a neurotrophin that binds with a high affinity to the receptor tyrosine kinase TrkA to stimulate neurite growth and differentiation. It also binds to the neurotrophin receptor p75NTR, which initiates apoptosis. NGF is primarily translated in its precursor form, proNGF. ProNGF makes up the majority of the NGF found in the central nervous system. After translation, proNGF is processed in the trans-Golgi network by proteases that interact with specific basic sequences and cleave proNGF to form mature NGF. Mature NGF has traditionally been considered the biologically active form of NGF. Previous studies demonstrated that the main biological function of proNGF is high affinity binding to the p75NTR receptor, which initiates apoptosis. In this study, they refute the claim that proNGF is inactive as a precursor except to stimulate cell death. Rather, they show that proNGF has functions similar to those typically attributed to NGF.

To assess the biological activity of proNGF, they developed a cleavage-resistant proNGF construct (proNGF (R-1G) ). To express cleavage-resistant proNGF, they inserted the mutated proNGF DNA into a baculovirus cell. They infected insect Sf9 cells with the baculovirus containing the cleavage-resistant proNGF and amplified it to obtain the secreted protein. They also obtained wild-type, unaltered baculoviruses and wild-type NGF inserted in baculoviruses to use as negative and positive controls, respectively. They took the protein-rich medium and tested whether any biological activity was apparent when proNGF (R-1G) replaced mature NGF in various cells. For example, in mouse superior cervical ganglion (SCG) neurons, neurite outgrowth and survival were assayed. Neurite outgrowth was also measured in PC12 cells. Finally, they analyzed proNGF (R-1G)’s ability to bind to the TrkA receptor and activate the mitogen-activated protein (MAP) kinase pathway in PC12 cells and NIH3T3-TrkA cells.

They determined that pro-NGF (R-1G) stimulates neurite outgrowth in SCG and PC12 cells, though the outgrowth was one-fifth less than outgrowth due to mature NGF. They also demonstrated that pro-NGF (R-1G) binds to the TrkA receptor, which would initiate the cell survival and differentiation pathway. Furthermore, they showed that pro-NGF (R-1G) promotes phosphorylation of TrkA in PC12 cells and in NIH3T3 cells. NIH3T3-TrkA cells do not have the second NGF receptor, p75NTR, proving that proNGF has a biological function apart from binding to the pro-apoptotic p75NTR receptor.

In conclusion, proNGF acts as a neurotrophin by promoting cell survival and differentiation. It functions similarly to mature NGF, though it is less active. The difference in rate may serve to mediate neurotrophic activity. If this is the case, proNGF would be cleaved to mature NGF when neurons need to rapidly grow and differentiate.

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Drosophila Neurotrophins Reveal a Common Mechanism for Nervous System Formation

Presented by Felix Camacho

Research and studies on brain development and the nervous system have mainly been based on vertebrates. However, little is known about the trophic interaction in the nervous system of invertebrate animals simply because evidence has been overlooked. Neurotrophins (NT) are a major class of secreted signaling molecules that promotes neuronal survival in vertebrates. NT links the nervous system structure and function, and is responsible for axonal elaborations and long-term potentiation, among other functions.

Previous studies have linked the Drosophila protein Spatzle (Spz) to NGF in the human genome, but have remained controversial. An initial BLAST method was performed to compare sequenced genomes of humans and Drosophila, but failed to account for the complete Drosophila genome. Zhu and his colleagues set out to investigate if Drosophila had any NT in the insect’s nervous system. They used TBLASTN and PSI-BLAST which are specific in detecting distantly related sequences. A FUGUE test was used to identify distantly related proteins.

From their experiments, they were able to identify a neurotrophic factor, DNT1, in Drosophila. DNT1 fit the criteria of a NT (expression in target cells, promotes neuronal survival, and enable targeting and selectivity) based on the observations of different DNT1 mutant phenotypes. They furthered their experiments by identifying Spz and DNT2 as part of a NT superfamily in Drosophila by performing more tests involving varying DNT1/Spz/DNT2 mutant combinations.

Based on their findings, it is now likely that neurotrophins were present in a common ancestor of bilateral organisms. These neurotrophins duplicated independently in vertebrates and invertebrates. It was likely so as NTs were found retained in organisms with a brain and a centralized nervous system. These finding are important as Drosophila can be linked again as a strong model organism that is helpful in studying human genome sequencing and diseases.

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