"Read More About It" Pages - Biology 376 - Fall 2008

Targeted expression of a human pituitary tumor-derived isoform of FGF receptor-4 recapitulates pituitary tumorigenesis

Presented by Ali Barry

The occurrence of pituitary gland tumors are becoming more and more prevalence in this day and age. These types of tumors are only of the many thousands of tumors that have been characterized but affect 20% of the population. The complete reason for the generation of these tumors is unknown but recent articles have helped to shed light on a certain part of this quandary. This paper examines of these parts: the tumor-derived, N-terminally truncated isoform of the FGF 4 Receptor named ptd-FGF4. Within the pituitary gland, there are several growth factors that regulate hormone production and are believe to also regulate pituitary growth. The FGF family have very important functions in many development processes: mesoderm induction, anterior-posterior patterning, limb formation, neural induction and brain development. The authors of this paper examined FGF1-3 looking for gross alterations that would implicate these tumors but it wasn't until they examined the structure and found the FGF4 isoform. The aim of this paper is to implicate and characterize the expression of this ptd-FGF4 in pituitary adenomas.

Several tests were done to complete this characterization of ptd-FGF4. First, tissues from pituitary adenomas were extracted and fixed for histological and immunochemical analysis. A normal FGF4 was used from a NIH 3T3 cell line, which is a standard laboratory fibroblast chemical line. The positive control used was from a human breast cancer cell line which also expresses FGF4. Using these experimentals and controls, the authors went on to test this newly-found isoform of FGF4 using methods that assessed the variations of function that make this form tumorigenic. One of the most revealing experiments done outlined the activity of the phosphostyrosine kinase, an enzyme that gets phosphorylated and is essential in signal transduction for enzyme regulation and is a main component of the FGF signaling pathway. Thus, this test is significant because it shows higher tumorigenic potential if there is constituitive phosphorylation of tyrosine kinase in this new form of FGF4 with or without the presence of FGF-1, as shown in transgenic mice.

In conclusion, this paper introduces the novel truncated isoform of FGF4, named ptd-FGF4, and through assesment of its characteristics has been deemed tumorigenic. Since it utilizes ligand-independence for tyrosine phosphorylation, it can be constituitively phosphorylated and thereby implicating the pituitary adenomas.

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Genetic Interactions between Brca1 and Gadd45a in centrosome duplication, genetic stability, and neural tube closure

Presented by Megan Lowers

Genetic interactions such as centrosome duplication, genetic stability, and neural tube closure between Brca1 and Gadd45a are shown to involve other genes and proteins including that of BAX, BCL-2, p53, and NEK2. Brca1 is a tumor suppressor gene which maintains genomic integrity to prevent uncontrolled proliferation. If Brca1 is mutated, it comes with a significant increase for developing breast cancer. The Brca1 protein is involved in DNA damage repair and transcriptional regulation as well as other functions. Gadd45a is a gene whose expression is regulated by two major tumor suppressor proteins, p53 and Brca1, involved in there mutated function to cause breast cancer. In addition, Gadd45a acts primarily in control of the G2/M cell-cycle transition, apoptosis, and DNA repair.

In order to observe the genetic interactions between Brca1 and Gadd45a, a few experiments were done to see the affect on centrosome duplication, genetic stability, and neural tube closure. Cells usually contain one or two centrosomes depending on their phases in the cell cycle. If the process is dysregulated, it can cause centrosome malformations. In the centrosome duplication experiment, they produced mice by crossing each mutant, Brca1Ä11/Ä11 and Gadd45a-/- (which from a previous paper were shown to suffer centrosome amplification). They found that the embryos were exencephalic (the neural tube was open) and that they showed a high level of apoptosis due to altered levels of BAX, BCL-2, and p53. The likely cause of these outcomes is genetic instability. For example, the increase in apoptosis in adults is associated with breast and ovarian cancer, whereas, when apoptosis is controlled it is important to maintain genetic stability. P53 plays an important role in getting rid of the cells with genetic damage.

In another experiment, Wang and colleagues used RNA interference to knockdown the expression of genes. In doing this they found that the suppression of Brca1 decreased NEK2, which is a cell cycle regulated protein kinase, more in Gadd45a-/- than in the wild-type cells. It was also observed that an overexpression of Brca1 and/or Gadd45a up-regulated transcription of NEK2. If NEK2 is overexpressed in the Brca1 small interfering RNA-treated wildtype and Gadd45a-/-, it was observed that the cells repressed abnormal centrosome amplification.

Conclusions: The absence of Gadd45a accelerates embryonic lethality of Brca1Ä11/Ä11 embryos. The Brca1Ä11/Ä11 and Gadd45a-/- embryos display exencephaly because of failed closure of the anterior neural tube. In addition, the Brca1Ä11/Ä11 and Gadd45a-/- embryos exhibit increased apoptosis. Both Brca1 and Gadd45a double deficiency activates a p53 mediated apoptotic pathway as well as decreasing NEK2 levels. Also, the expression of Brca1 and Gadd45a up-regulates NEK2 expression. Overall, this paper shows that both Brca1 and Gadd45a are involved in maintaining NEK2 expression, while down-regulation of NEK2 in mutant cells could be a cause of centrosome amplification. This amplification may lead to genetic instability, increased apoptosis, and other abnormal consequences.

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Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas

Presented by Amy Hernandez

Cellular senescence,is a growth-arrest program that limits the lifespan of mammalian cells and prevents unlimited cell proliferation. It can be triggered by multiple mechanisms such as telomere shortening and DNA damage. This process, first observed in cell culture, is now confirmed by in vivo evidence to be a vital mechanism that prevents the malignant progression of many tumours.

The tumour suppressor gene p53 is a common gene found to restrict proliferation of any DNA damage in the cell senescence program. p53 mutations are common in human liver cancers and its considered to be highly aggressive and resistant to nonsurgical therapies. Reversed RNAi was used in this study in order to control p53 in chimaeric liver cancer mouse models and to determine the requirement for p53 loss in the maintenance of liver carcinomas. Embryonic liver cells (hepatoblasts) were transduced with retroviruses expressing oncogenic ras (HrasV12), tetracycline transactivator protein (tTa) and a tet responsive p53 miR30 design short hairpin RNA (shRNA). In vivo imaging was performed:oncogenic ras coexpressed GFP. After the onset of liver tumours, p53 expression was restored by injecting doxycycline(Dox), a common treatment used for infections. One experiment done showed p53 expression with or without Dox. Result: Dox treatment turns off the conditional miR30 based p53 shRNA. The reactivation of p53 led to tumour regression.

Another experiment involved subcutaneous tumours derived from ras-transformed liver progenitor cells with tet-off shRNA (TRE.shp53) or a non-regulatable shRNA (MLS.shp53)that were grown in nude mice. Result:Brief reactivation of p53 is sufficient to suppress colony formation and regress subcutaneous tumours. In order to determine whether or not the primary response to p53 is apoptosis, Haematoxylin and eosin (H&E) immunohistochemical staining for apoptotic cells (TUNEL and Caspase 3 staining) and proliferating cells (Ki67 staining) of liver tumours before (p53 off) and after Dox treatment (p53 on)were performed. Result:Tumours showed histopathology of human hepatocellular and cholangiocellular carcinoma. p53 promotes cellular senescence, to show this B-gal activity and senescence markers were observed. Hepatocarcinomas expressing oncogenic ras showed clear signs of senescence following the reactivation of p53 in vivo. Examinations of tumours were observed at different times following reactivation revealing a progressive inflammatory reaction. An immune inflitration was revealed in regressing tumours following p53 activation. In addition, increased expression of chemokines and adhesion molecules in senescent tumours (in vivo) and cultured liver progenitor cells (in vitro)were observed. Subcutaneous hepatocarcinomas were treated with Dox in order to induce regression and to determine if innate immune cells were required for tumour clearance. Using macrophage toxin;gadolium, and neutralizing antibodies these treatments delayed tumour regression thus confirming that components in innate immune system were actively involved in tumour clearance.

Conclusions of the paper: The requirement for p53 loss in the maintenance of such carcinomas,p53 can induce tumour involution through a process that, once activated, seems irreversible. The primary response to p53—senescence,a pulse of p53 activity was sufficient to trigger senescence in vivo. The other response to p53—innate immune response, showed multiple components of the innate immune system can infiltrate the tumours following p53 reactivation. The induction of cellular senescence and the evoked immune attack cooperate to promote tumour clearance.

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p19Arf Suppresses Growth, Progression, and Metastasis of Hras-Driven Carcinomas through p53-Dependent and -Independent Pathways

Presented by Michelle Juanengo

p53 is a tumor suppressor gene that is most mutated in cancers. It’s also a transcription factor that turns on a cyclin-dependent kinase (cdk) inhibitor p16. (Cdk inhibitors block entry into S phase of the cell cycle.) p16 (INK4a) suppresses cell growth and is linked to another tumor suppressor p19 (ARF = Alternative Reading Frame). p19Arf stabilizes p53 by binding to Mdm2, which inhibits p53 degradation. When p53 is activated tumor progression is suppressed. Both p16 and p19 are encoded on the INK4a-ARF locus and the primary focus on this paper targets tumor suppression of p19 though p53 pathways.

Ras is an intracellular protein, more specifically a GTP binding protein that is mutated in many cancers. This commonly mutated oncogene plays a role in multiple signaling pathways which can lead to formation of tumors, more specifically carcinomas (malignant tumors) and cancer cell metastasis. The mutation in Ras used in this experiment is seen in the family member Hras, which promotes Hras-driven carcinomas.

The relations between Ras, p19Arf, and p53 are tested in three types of mice: wildtype, p19Arf- deficient, and p53-deficient mice. Carcinogens such as DMBA/TPA were injected into the mice to induce tumors. Papilloma (benign tumors) growth rate, the malignant conversion of papillomas to carcinomas (malignant tumors), and metastasis of carcinomas were compared in the different types of mice. The progression of squamous cell carcinomas (SCCs) caused by increased Ras acitivity shows that expression of Ras induces p19Arf expression, which as previously stated, stimulates p53. Activated p19Arf inhibits Hras tumors through a p53 independent pathway. However, p19Arf also inhibits progression of malignant tumors and metastasis through a p53 dependent pathway. Therefore p19Arf regulation of tumors can be both independent and dependent of p53.

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The Human Papillomavirus E6 Oncogene Dysregulates the Cell Cycle and Contributes to Cervical Cancinogenesis through Two Independent Activities

Presented by Tracy Mrowczynski

Cervical cancer is the second most common type of cancer in women and the leading cause of death of women from cancer worldwide. Among this cancer, most are associated with the human papillomavirus (HPV) and about half are caused by the HPV16 strain. Two previously identified HPV16 viral oncogenes include E7 and E6. Prior studies have shown that E7 and E6/E7 transgenic mice displayed high incidence of dysplasia over a 6 month period. However, E6 transgenic mice only exhibited low grade dysplasia. This paper studied the effects of E6 over a 9 month period by looking at various biomarkers to further examine oncogenic potential of E6.

The study used two of transgenic E6 mutants: K14E6I128T and K14E6delta146-151. The K14E6I128T mutant, which had a single amino acid mutation, has a reduced ability to bind to a-helix partners and the K14E6delta146-151 mutant, which had several amino acid deletions, has a reduced ability to bind to PDZ partners. For 9 months, these mutants and K14E6WT transgenic mice were treated with high dose estrogen, which is a cofactor in cervical carcinogensis.

Overall, 41% of the K14E6WT mice developed cancer while only 6.7% of the nontransgenic mice did. These results are important because it shows that E6 can cause cancer in absence of E7. Tumors from the K14E6I128T mutant occurred less and were smaller than K14E6WT and K14E6delta146-151 mice illustrating the importance of a particular amino acid in the protein in promoting cancer. E6 had a 1.5 times greater presence in the K14E6delta146-151 mice cells than the K14E6I128T and the K14E6WT mice cells. These mice also had the highest cancer incidence of any group and the increase in hyperplasia is because of an increase in DNA synthesis due to interactions with PDZ partners. E6 transgenic mice inactived p53 and upregulated cyclin E, though the effects were lesser than in E7 mice. E6, however, did alter p16 expression (a cdk inhibitor that acts like p21) via an interaction of E6 and ά-helix partner and this was different from E7 oncogene interactions. In general, E6 exhibit oncogene potential, though it is weaker than E7, and it contributes of cervical carcinogensis through interactions with ά-helix and PDZ partners.

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ARF functions as a melanoma tumor suppressor by inducing p53-independent senescence

Presented by Kathryn Petersen

Many of the cancer therapies involve targeting the p53 gene due to high consistencies in cancer patients and inactivated p53 pathways. However in the case of melanoma, it has been observed that the p53 pathway is not inactivated in a majority of individuals with melanoma. For this reason, it is hypothesized that there must be some other defect that causes melanoma. Melanoma is often associated with insufficiencies of ARF rather than p53. ARF is a tumor suppressor that functions independently of p53 and associates with proteins such as E2F1, Myc, NFkB and CtBP.

The paper performed a variety of experiments aimed at determining whether the hypothesis that ARF is a major cause of melanoma and it does so independently of p53. One of the first findings was that there are no significant differences between the age of melanoma onset in Arf (+/-) and Arf (-/-) transgenic mice. Further experiments showed that ARF works independently of p53 because mice deficient of ARF but with functional p53 genes still developed melanoma after a short time. The combination of many of the experiments discussed in this paper emphasize the role of ARF in the regulation of senescence as they demonstrated that the melanocytes formed tumors easily in vivo when ARF was damaged regardless of the functionality of p53.

The implications of this paper could prove to be beneficial for the progression of cancer treatments. If further studies prove the importance of ARF rather than p53, cancer treatments could be altered to better treat this dysfunction.

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p53 Suppresses the Self-Renewal of Adult Neural Stem Cells

Emily Young

One of the main characteristics of stem cells is their capacity for continuous self-renewal. These characteristics can be compared to fast expansion of cancer cells. This process of self-renewal can be regulated by modification of many cell processes, including apoptosis, cell proliferation and cell differentiation. p53, a tumor suppressor gene, is thought to be a major gene involved in regulation of apoptosis and in turn cell proliferation. This function of p53 is important in studying its effects on the self-renewal of neural stem cells, which have the potential to become cancerous. The basis of this experiment was to study the effects of the deletion of the p53 gene on the continuous growth of neural stem cells.

In order to test the effects of p53, two different genotypes of mice were used. Those with the p53 -/- genotype and those with the p53 +/+ genotype. The expression of p53 was first observed in the wildtype mice and found to be most prevalent in the lateral ventricle wall, but less prevalent in the subventricular region. p53's effect was also studied by observing the number of proliferating cells, number of apoptotic cells, and size and density of neurospheres in each genotype. Western Blot, PCR, TUNEL, and Microarray were used to analyze the results.

Overall, the results supported the theory that p53 is a negative regulator of stem cell self-renewal. When p-53 was absent in the mice, cell proliferation increased and the amount of apoptotic cells decreased. The number of neurospheres and their volume also increased once p53 was deleted. The self-capacity of these cells also increased, but the multipotency remained the same. This illustrates that p53 may not have a role in controlling neural differentiation. All of these results support the fact that p53 is a negative regulator of cell proliferation.

The results of this study can be related back to cancerous cells because p53 is commonly mutated in the majority of tumors found in the human body. These mutations result in p53 not being transcribed and therefore increased cell proliferation occurs. This may also increase the probability of acquiring more mutations which in turn could evolve into cancerous cells. This emphasizes the importance of p53 and other tumor suppressor genes in order to maintain normal cell growth and death.

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Suppression of Tumorigenesis by the p53 Target PUMA

Presented by Rebecca Cisneros

p53 tumor suppressor is a transcription factor that has many roles in cells such as regulating cell cycle check points, DNA repair, and apoptosis. Disruption of any of these activities could explain the impact of p53 mutations. Attempts have been made to find out if certain p53 effectors (target genes) can mimic p53 loss during tumorigenesis. PUMA, a proapoptotic member of the bcl2 family, is a p53 target gene that modulates Bax to release cytochrome c which then initiates the apoptotic cascade. Since PUMA can be an important mediator of p53 dependent apoptosis, this paper uses RNA interference to see if PUMA is a potential tumor suppressor by seeing if it can mimic p53 activities.

First RNA interference is tested to see if it can stably suppress PUMA expression. PUMA short hairpin RNAs (shRNA) that target specific sequences of PUMA were generated and introduced to mouse embryonic fibroblasts (MEFs) using retroviral vectors. The PUMA shRNAs were successful in suppressing PUMA expression in the MEF’s just like p53 shRNAs.

PUMA was then tested to see if it affects p53 dependent cell cycle arrest when it is suppressed. MEFs with PUMA shRNAs and p53 shRNAs were blasted with gamma radiation and PUMA shRNAs still retained cell cycle checkpoints while p53shRNAs did not. This shows that PUMA does not affect p53 dependent cell cycle arrest. Since p53 can cooperate with E1A / ras oncogenes and just ras alone to promote oncogenic transformation, PUMA was tested to see if it could do the same. In the cells that co- expressed E1A/ras, Puma shRNAs induced transformation which was seen by rapidly progressing tumors. However no transformation occurred in the MEFs that had ras alone. This shows that PUMA suppression can mimic p53 loss by some oncogene combinations but not others.

In previous tests, p53 suppression became known to accelerate Eì-myc lymphomagenesis. Again, PUMA suppression was tested to see if it could do the same. Homeotic stem cells that encoded a PUMA shRNA we put into mice and these mice developed lymphomas just as quick as the p53 loss effect. These results show that PUMA can function as a potent tumor suppressor because it’s suppression can mimic most of the effects of p53 loss. Overall this paper shows that p53s tumor suppression activities are context dependent because apoptosis disruption by PUMA loss is enough to promote tumorigenesis in E1A/ras transformation and myc-induced lymphomagenesis settings whereas cell-cycle checkpoints and genomic instability are byproducts of p53 suppression.

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Direct Activation of Bax by p53 Mediates Mitochondrial Membrane Permeabilization and Apoptosis

Presented by Brett Vincent

When normal mammalian cells are subjected to various stress signals, such as radiation or DNA damage, the tumor suppressor p53 is activated. Upon activation, p53 functions as a transcription factor, promoting the expression of a number of genes which function in the pathway leading to apoptosis (programmed cell death.) However, recent studies have suggested that p53 may also have the ability to induce apoptosis independent of transcription. That is, cytosolic p53 (absent from the nucleus) directly ‘triggers’ the Bax protein, enabling its permeabilization of the mitochondrial membrane and the release of cytochrome c.

This particular study explored these claims, testing whether or not endogenous p53 can engage the apoptotic pathway directly from the cytoplasm and independent of transcription. The first part of the experiment implemented the use of oncogene-expressing MEF (mouse embryonic fibroblasts) deficient in p53 or Bax and their respective wild-type controls. Upon investigation, they found that when subjected to UV radiation, the p53-deficient MEF as well as Bax-deficient MEF did not undergo apoptosis, however wild types, which expressed both p53 and Bax, did. This experiment confirmed that p53 induced apoptosis is dependent on Bax.

In a second experiment, researchers were seeking to find out whether or not p53 itself is capable of inducing Bax permeabilization into the mitochondrial membrane. This was carried out by separately incubating Bax with mitochondria; p53 with mitochondria; and the combination Bax and p53 with mitochondria and later noting which of these combinations lead to the release of cytochrome c. They found that alone, p53 and Bax do not induce the release of cytochrome c from mitochondria. However, when Bax and p53 were incubated together with mitochondria, cytochrome c was released. This leads to the conclusion that p53 somehow aids in Bax permeabilization of the mitochondrial membrane.

Lastly, it is understood that Bcl-2 family proteins Bid and Bim can serve to induce a conformation change on Bax enabling its mitochondrial permeabilization. However, Bid and Bim also bind to apoptotic repressor proteins, Bcl-2 and Bcl-xL, to aid in the blocking of apoptosis by sequestering proapoptotic Bcl-2 members. p53 has also been reported to bind to Bcl-2 and Bcl-xL, but the reasons as to why were unclear. An experiment was conducted to determine what function p53 serves in binding to these apoptotic repressors and it was concluded that p53 displaced Bid and Bax sequestered by Bcl-xL. The following experiments performed show us that as a result of UV radiation and DNA damage, p53 proves to be capable of inducing apoptosis by direct activation of Bax. In addition p53 has displayed capabilities analogous to BH3-only proteins, aiding in Bax permeabilization of mitochondria by blocking apoptotic repressor proteins.

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PDGF signalling controls the migration of mesoderm cells during chick gastrulation by regulating N-cadherin expression

Presented by Olivia Krzmarzick

Epithelial-to-mesenchymal transition or EMT occurs during invagination in early chick development. The cells that are in the epiblast go toward the primitive streak and then become different mesodermal structures such as somites and lateral plate mesoderm. PDGF is important in signaling this mesoderm cell movement. The receptors (PDGFR) are found in the mesoderm while the ligands (i.e. PDGFA) are located in the ectoderm. If the receptor or ligand is inhibited in any way, there are major consequences in the development of the organism, such as spina bifida. Since there are two different receptors (PDGFRa and PDGFRb) that exist with many different ligands, the information on this signaling system is not well known. By studying PDGFRa along with the main ligand, PDGFA, control of the migration of mesoderm cells is demonstrated. N-cadherin expression was also found to be controlled by PDGF signaling.

After experiments, Pdgfa was found to be present in the epiblast on both sides of the primitive streak. When PDGFRa was blocked with a dominant-negative PDGFRa, mesoderm cells did not migrate to the primitive streak as in a normally developing chick. Also when recombinant PDGFRa-Fc protein minimized the ligands, the same migration was also inhibited. Similar experiments also demonstrated that PDGF navigates the migration of these mesoderm cells. Some of the results showed a decrease in development when the ligand or receptor was blocked, but there was also a decrease in N-cadherin expression. N-cadherin expression is controlled by the PI3 kinase-dependent signaling pathway in the mesoderm cells, which guides migration. There are many different mechanisms that must work together in order to make sure that normal development takes place.

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p53- and drug-induced apoptotic responses mediated by BH3-only proteins Puma and Noxa

Presented by Sean Kenmore

The tumor suppressor gene p53 is known to prevent tumor development inducing the expression of the cdk-1 inhibitor p21. This effectively prevents the damaged cell from entering S phase of the cell cycle, which prevents the cell from dividing. p53 also has over 16 different target genes believed to mediate apoptosis, although how they trigger programmed cell death is still unknown. Two possible proteins that are p53 gene targets may be critical to activating apoptosis, the BH3-only (also known as BH3) proteins Puma and Noxa. These proteins initiate apoptosis by binding to Bcl-2 (apoptosis suppressor), leading to the degradation of the cell by proteases. It is also believed that these two proteins may be involved in the p53-independent pathway to apoptosis, because cell death will be prevented if Bcl-2 is over expressed.

In order to test the effect of these proteins, mutant strains of mice were created in which Noxa or Puma was deleted respectively. This was done by deleting important sequences in of these genes in embryonic stem cells. Multiple tests were used to ensure that these mutants were actually homozygous deletions, including: reverse transcription-polymerase chain reaction to ensure a smaller gene sequence, and Northern blots on various tissues to ensure the missing presence of the genes in different tissues. It was also determined that these deletion mutants developed normally in terms of frequency, appearance, organ weights, cellular compositions, and hemopoietic organs.

Gamma radiation and the topoisomerase inhibitor etoposide were used to determine the effects of p53-dependent apoptosis by causing DNA damage. To test p53-independent apoptosis, different chemicals were used to initiate apoptosis. Noxa deletions were assessed using thymocytes, pre-B cells, mature B and T cells, and mouse embryo fibroblasts (MEFs), while Puma deletions were assessed in thymocytes, other lymphocytes, and MEFs.

It appears that both Puma and Noxa play an important role in the p53-mediated pathways of apoptosis, as cells with both of these deletions showed significant rates of survival when exposed to DNA damage. However, in the p53-independent pathways, it appears that only Puma has a significant role in programmed cell death, as many of the cells continued to survive despite the different methods used to try and induce apoptosis. Both of the BH3-only proteins show significant involvement in the apoptotic pathway, with Puma being the more prominent of the two molecules.

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Haploinsufficiency of Bcl-x leads to male-specific defects in fetal germ cells: differential regulation of germ cell apoptosis between the sexes

Presented by Sarah Manahan

Apoptosis is both a normal, and neccessary process, occurring during development. It is a process carefully regulated by the body via apoptotic promoting and preventing factors. Apoptosis occurs in much the same way in both the somatic and germ cells, and misregulation in either may lead to a devastating phenotype.

Bcl-x knockout mice were used in this experiment to identify the differences in germ cell apoptosis between the sexes. A homozygous knockout of Bcl-x proved to be lethal in mice early in development. However, heterozygous mice were normal in external appearance, differing by sex in the germ cell effects of the partial gene knockout. Heterozygous females do not show abnormalities in germ cells until later in their reproductive life, while the effect on males incurs at a much earlier stage, during development, resulting in reduced fertility.

Bcl-x is an apoptosis suppressor molecule, disrupting Bax, which promotes apoptosis. The homozygous lethal mutant mice are viable until embryonic day 13.5. This is the same day in which regulation of germ cell apoptosis differentiates by sex in heterozygous mutants. The significance of this day is that this is also when sex differentiation occurs in the embyro, leading the developing mouse to follow different apoptotic pathways depending on sex. On day 12.5 of development, the germ cell count does not significantly vary between wild type, heterozygous, and homozygous mice. It is only after day 13.5 do the cell counts vary by sex, and in the case of the males, by genotype. Therefore, Bcl-x expression is not needed for germ cell sustainability prior to day 13.5. It is during this stage in development that Bcl-x is crucial to germ cell viability, with importance varying by sex.

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Nov. 19, 2008 Presentation

Peroxynitrite Mediates Retinal Neurodegeneration by Inhibiting Nerve Growth Factor Survival Signaling in Experimental and Human Diabetes

Presented by Ryan Brown

Neural [Nerve] Growth Factor or NGF is a family of proteins that induce the survival and proliferation of target neurons. NGF is a family of proteins also known as neurotrophins. NGF binds to the TrkA receptor (p13K/Akt cascade pathway), which leads to a neuronal survival signal. If this pathway is interrupted it may drastically alter neurons in the area. NGF also binds (but with less affinity) to the P75NTR receptor, which causes a cascade leading to apoptosis (through the JNK p38 pathway).

This paper shows evidence that the presence of peroxynitrite impairs NGF ability to signal neural survival, by nitrating the TrkA receptor and stopping this signaling pathway. At the same time peroxynitrite enhances p75NTR expression in cells lacking TrkA activity or expression, causing apoptosis.

Individuals with Diabetes have a high concentration of peroxynitrite in their peripheral nervous system. One cause of diabetic retinopathy is hypothesized to occur via this NGF TrkA pathway. This paper looked at both human eyes in vitro and diabetes-induced rat eyes in vivo to show how the presence of peroxynitrite can cause neuronal death. Interestingly however, the addition of FeTPPS, which breaks down peroxynitrite, can stop neural death and restore the pathway.

The over-expression of both NO and superoxides leads to the formation of peroxynitrite. Normally NO alone will not lead to the production of ONOO (peroxynitrite). However, diabetics also have higher concentrations of superoxides (O2-). NO combines with superoxides to produce the peroxynitrite radical. By examining this pathway, which may lead to diabetic retinopathy, scientists may be closer to preventing the development of DR, which is the leading cause of blindness in working age adults.

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