"Read More About It" Pages - Biology 376 - Fall 2010

Dec. 10, 2010 Presentations

Oncogenic activation of the RAS/RAF signaling pathway impairs the response of metastatic colorectal cancers to anti-epidermal growth factor receptor antibody therapies

Colorectal cancer, also called colon cancer, involves cancerous growths in the colon, rectum, and appendix. Worldwide, there more than 600,000 deaths due to this type of cancer. Additionally, it is the fourth most common form of cancer in the United States and the third leading cause of cancer-related deaths in Western society today (World Health Organization). Today, valuable treatments using epidermal growth factor receptor (EGFR) inhibitors have emerged. EGFR is a transmembrane tyrosine kinase receptor involved in the proliferation and survival of cancer cells. Therefore, inhibition of this receptor is a common target in cancer treatment. However, some cancers have developed resistance to this treatment through various means; one of which is oncogenic activation of the RAS/RAF signaling pathway.

Using mutational analysis and CRIB assays, Benvenuti et al., showed mutations that improperly activate the RAS/RAF signaling pathway may be prognostic indicators in metastatic colorectal cancer patients. The researchers also found that activation of the RAS pathway via an activated K-RAS allele inhibits the therapeutic effects of EGFR inhibitors. Yet, the researchers also found that in cancer cells with highly active RAS, inhibition of the mitogen activated protein kinase (MAPK) signaling pathway may facilitate various EGFR treatments.

This is significant because these results have several implications. One is that patients with colorectal cancer carrying mutated K-RAS are not likely to experience beneficial therapeutic treatments via EGFR inhibitors. However, this study found some of the K-RAS mutations are actually compatible with EGFR treatments and therefore, patients with mutated K-RAS should not be hastily excluded from EGFR treatments. The second implication is this research supports the idea for multitherapies involving both EGFR and MAPK inhibitors for treating patients with metastatic colorectal cancer.

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The KLF4 tumour suppressor is a transcriptional repressor of p53 that acts as a context-dependent oncogene

Kruppel-like factor 4 (KLF4) is a transcription factor that can act in two ways: as a repressor and as an activator involved in cell-cycle regulation and cellular differentiation. Therefore it can act as a gene regulator to inhibit cell proliferation. KLF4 has been found to act as a tumor suppressor because it acts as an inhibitor of cellular proliferation. KLF4 has also been found to be associated with cancer. When levels of KLF4 are increased carcinomas are likely to occur. So this means that while KLF4 can act as a tumor suppressor, if it is absent or over-expressed it can lead to tumor formation.

Many experiments were done to find how KLF4 acts as a tumor suppressor and how it acts as an oncogene. KFL4 was found to bypass RAS induced senescence meaning that KLF4 can bypass the RAS pathway to induce cell senescence. This would further show that even if the RAS proto-oncogene is mutated into an oncogene, which would promote cell proliferation and continuation of the cell cycle, KLF4 would still cause senescence to occur.

KLF4 acts as an oncogene because of its relation to p53 and p21. KLF4 has opposite effects on p53 and p21. KLF4 leads to an increase in the levels of the cyclin-kinase inhibitor p21 but leads to a decrease in the levels of the tumor suppressor p53. p53 has been shown to control the transformation of the proto-oncogene RAS to its oncogene; so when KLF4 is present it suppresses p53 it allows for the bypass of RAS induced senescence and through this suppression DNA damaged induced apoptosis.

KLF4 also interacts with p21 in that KLF4 induces the levels of p21. In the absence of p21, KLF4 changed from a tumor suppressor protein into a oncoprotein and contribute to cellular transformation. Cyclin-D1, a target of RAS, is essential to proliferative collaboration between KLF4 and RAS, and when it is depleted RAS and KLF4 induced proliferation is impaired. In breast cancer cells KLF4 plays a vital role in breast cancer cell survival because it suppresses p53, which would case breast cancer cells to die. The suppression of p53 in breast cancer cells is dose-dependent.

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p16INK4a Modulates p53 in Primary Human Mammary Epithelial Cells

most commonly inactivated tumor suppression genes in various tumors, including breast cancers are p53 and p16INK4a (p16). Inactivation of these genes can occur by various mutations including, deletion, point mutations, and epigenetic silencing. p16 genes code for a cyclin-dependent kinase inhibitor, which initiates G1-phase cell cycle arrest when it detects DNA damage, oncogenic stress, and oxidative stress. p53 codes for a multifunctional protein that can work as a transcription factor to initiate cell cycle arrest, or apoptosis in response to the same factors as p16. p53 is induced by post-translational modifications that stabilize its protein and modulate its binding to other proteins and DNA. Although these genes are induced under the same conditions, it has been found that the gene inhibition may occur at differing times.

In breast carcinoma, it has been hypothesized that p16 inactivation occurs very early on in carcinogenesis, which can increase p53 activity which later promotes its inactivation. This hypothesis was tested using primary human mammary epithelial cells (HMEC) to examine the interactions between the p53 and p16 inactivation pathways.

This study found that HMEC demonstrate an inverse relationship between p16 and p53 protein levels by isolating and culturing primary HMEC and examining p16, p53, and the p53 target gene, p21 protein levels by Western blots. They then examined the functionality of the newly elevated p53 protein levels by infecting the HMEC with a retrovirus to inhibit p53 function. They learned that the inverse regulation of p53 by p16 in turn regulates p53-dependent proliferation. Stress responses to DNA damage through radiation were also found to be regulated by p16 modulation of p53 by using ionizing radiation on HMEC with inhibited p16 genes. In addition, p16 expression was found to decrease p53 protein stability by inducing a proteasome mechanism. Finally, the degradation of p53 protein was found to occur by a retinoblastoma pathway in a cell type specific manner.

The findings of this study are important in that they depict a new way by which carcinogenesis can occur and may provide a mechanism for a sequence of genetic changes that occur in many tumors. No other studies have found such an antagonistic relationship between these two crucial tumor suppressor genes, but such information can provide a new direction for therapeutic targets.

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Glioma oncoprotein Bcl2L12 inhibits the p53 tumor suppressor

Gliomablastoma multiforme (GBM) is a malignant brain tumor which runs an aggressive course and is therapy-resistant. This is the most severe grade of brain tumor and is characterized by rapid tumor cell growth, apoptotic resistance, extensive necrosis, and angiogenesis. Patients with GBM do not often live more than 12 to 18 months after diagnosis. Bcl2L12 is a member of the Bcl-2 family proteins which are known for their role in promoting cell survival. Bcl-2 proteins are known to interact with p53. However, Bcl2L12 is an atypical member of this family as seen by its prominent localization in the nucleus as opposed to the normal mitochondrial/cytoplasmic distribution. Furthermore, Bcl2L12 was previously identified by Stegh and his colleagues as an anti-apoptoptic and pro-necrotic oncoprotein and a regulator of cell death in glial cells. They recently discovered that Bcl2L12 plays a role in glioma pathogenesis through inhibition of effector caspase-3 and caspase-7, which are two proteases that trigger the apoptotic process. Nearly all GBM tumors exhibit high expression of Bcl2L12, whereas in less severe astrocytomas and normal brain tissue expression is low or absent. This knowledge of Bcl2L12 led to the exploration of a potential link between the protein and p53, the tumor suppressor gene responsible for the transcript ional control of over 2500 genes involved in cancer-relevant processes such as cell cycle regulation.

The current paper presents novel data that further define the role of Bcl2L12 in glioma development with a focus on its physical and functional interaction with p53. Mouse embryonic fibroblasts (MEFs) were retrovirally transduced with either Bcl2L12 or the control pBabe and allowed to grow in culture. While the control cells showed reduced growth rates and growth arrest, the cells expressing Bcl2L12 continued to grow through over 35 passages. Despite the reduced senescence due to Bcl2L12 expression, there was no loss of p53 in the genomic DNA. In order to observe the affects of Bcl2L12 on p53 induced programmed cell death, the MEF cells were treated with doxorubicin (DOX), which is a DNA-damaging agent. The Bcl2L12 MEFs showed slightly less p53 expression. In addition, Bcl2L12 expression led to significantly greater inhibition of p53- dependent apoptosis in DNA-damaged cells in comparison to the controls.

Immunofluorescence microcrospy was one of several techniques used to determine whether there is a physical interaction between p53 and Bcl2L12. It was confirmed that colocalization occurs within the nucleus. In vitro assays showed that the Bcl2L12:p53 complex is relatively robust in comparison with other p53 interactions. The influence of Bcl2L12 on the transactivation potential of p53 was examined in order to confirm their suspected interaction. RT-PCR analyses provided evidence that Bcl2L12 blocks the transcription of several well-known p53 target genes that regulate the cell cycle and apoptosis, including p21, DR5, Noxa, cyclin G1, and Puma. However, this inhibitive activity was selective: Mdm2 expression was not affected by Bcl2L12. The authors conclude that Bcl2L12 selectively represses p53-dependent transcription by binding to target promoter sequences. The Bcl2L12:p53 axis is a potential target of drug therapies which aim to reactivate the growth arrest and apoptosis of cells which is prevented in cases of GBM. Inhibition of Bcl2L12 binding to p53 would allow for the key proapoptotic target genes of p53 to function normally and for proper regulation of the cell cycle.

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Senescence-Associated Secretory Phenotypes Reveal Cell-Nonautonomous Functions of Oncogenic RAS and the p53 Tumor Suppressor

Presented by Amelia Pedneault

Tumor growth is based upon the cell division that occurs in damaged cells. In this study, CoppŽ et. al (2008) discuss the role of cellular senescence in formation and suppression of tumors. This paradox is called antagonistic pleiotrophyÑan evolutionary theory of aging. Cellular senescence can function to block cell division and thus prevent damaged cells from undergoing proliferation, suppressing tumors. Their findings, however, also suggest that the effects of cell aging can be deleterious by formation of secretory phenotype that encourages tumor formation. This phenotype not only affects tumor cells, but may have an effect on nearby cells as well. The p53, p16INK4a/pRB, and RAS proteins regulate senescent responses in tumor suppressor pathways. Their pathways direct cells to die, proliferate, or pause growth.

Coppe et. al performed various tests on human senescent cells and examined effects both in culture and in vivo. Specifically, they began by looking at the proteins these cells secrete into their microenvironments. They found that senescent cells secrete inflammatory and immune-modulatory cytokines, chemokines, growth factors, shed surface molecules, and malignant factors. The secretion of these led to the discovery of what they titled the senescence-associated secretory phenotype (SASP), which describes a range of features. In this study, secretion levels of various SASP proteins were verified by ELISAs, and were found to increase, in comparison with a normal cell, up to ten fold.

They also found that normal fibroblasts, epithelial cells, and tumor cells submitted to genotoxic stress in culture (derived from various human donors) or chemotherapy (in an in vivo study) demonstrate SASPs. They developed modified antibody arrays which allowed identification of factors responsible for malignant pheotypes. It was noted that active mutant RAS proteins and loss of p53 function contribute to SASP. SASPs manipulated to have both of these characteristics developed a paracrine mechanism involving interleukins 6 and 8 that alter the microenvironment of the cells. This microenvironment alteration leads to invasiveness of the cells, which is how it may contribute to formation of tumors. It was also suggested that the cytokine receptors secreted by SASPs act as decoys and allow tumor cells to escape the immune systemÕs destructive function. And, by measuring mRNA levels via quantitative PCR, it was evident that tumors subjected to genotoxic stress contained high levels of p16INK4a, which also is commonly indicative of tumor proliferation.

To further understand the purpose of p53 in senescence, they inactivated p53 using genetic suppressor element peptide. By doing so, they found that senescence did not occur and a SASP did not develop. However, when forced to senesce by other factors (REP or RAS), the SASP was more exaggerated with no p53. This indicates that p53 is responsible for restraining development of a SASP. Thus, p53 prevents malignancy. Increased expression of RAS resulted in the oppositeÑand encouraged invasiveness of SASPs. It was found that many factors were upregulated in mRNA abundance, indicating that SASP factors are regulated transcriptionally. As previously stated, the study looked at antagonistic pleiotrophy. They concluded that senescence can be both beneficial and deleterious, as senescent cells contribute to both growth arrest of tumors and the secretory phenotype.

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Role of STAT3 in In Vitro Transformation Triggered by TRK Oncogenes

Presented by Sean Hurley

There are many factors that can lead to the development of cancer. One thing that has been found in many cancers is the presence of oncogenes. Many normal genes, often relate to cell division or death, are know as proto-oncogenes and a simple mutation can cause them to become oncogenes. Oncogenes contribute to the development of cancer because the proteins from the mutated genes can no longer be regulated the way that the normal protein would be. This leads to unregulated or abnormal cell division. This paper investigates the role of Signal Transducer and Activator of Transcription (STAT) 3 in the effects caused by the TRK (tyrosine kinase) oncogenes.

STAT 3 has been found to have various biological functions and be involved in processes such as cell proliferation and carcinogenesis. Other functions of STAT 3 include signal transduction, microtubule mediation, and transcription factor. All these functions make it no surprise that activated STAT 3 has been found in tumors of many various types of cancer. It has also been found that the inactivation of STAT 3 can inhibit some malignancies. Activated STAT 3 was found to have two roles in the TRK oncogenes by this study. These two roles are the decreased STAT 3 levels caused by the TRK oncoprotein activity are associated with morphological transformation in the cells and that residual STAT 3 transcriptional activity is required for cell growth.

First, STAT 3 activation or phosphorylation had to be shown to be caused by TRK oncogenes. Phosphorylation of STAT 3 promotes the transcription of target genes by dimerizing and translocating it to the nucleus. The phosphorylation of STAT 3 is done through the MAPK pathway to the affect of inhibiting this pathway was also examined. Treatment with a MEK inhibitor resulted in reduced STAT 3 phosphorylation. To test STAT 3 in vitro, different cells expressing TRK, TRK-T1, and TRK-T3 oncogenes were used.

After the confirmation that STAT 3 was activated by the TRK oncogenes, the affects of STAT 3 were examined. First, the morphological transformations observed in cells with TRK oncogene activity were connected to a reduction of STAT 3. When cells were treated with a TRK kinase inhibitor, STAT 3 levels were found to have increased and phenotypic changes were revised. Cytoplasmic STAT 3 was also shown to play a role in microtubule stabilization. This is due to interactions with the protein stathmin, which usually induces the disassembly of the microtubules. Second, the importance of STAT 3 on cell growth was determined by using a STAT 3 inhibitor. Cells treated with this inhibitor displayed a very significant reduction in cell growth over 24 and 48 hours. This demonstrates that the unregulated activation of STAT 3 can result in rapid cell growth.

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Protein tyrosine phosphatase receptor-type O (PTPRO) exhibits characteristics of a candidate tumor suppressor in human lung cancer

Presented by Brian Schneider

Protein tyrosine phosphotase receptor type O, or PTPRO, is examined as a candidate in tumor suppression. It was originally noted for having classical tumor suppressor characteristics in rat liver tumors. The study that I looked at involved similar findings in human lung cancers. PTPRO is important to study because it has been shown that its over expression aids in preventing cell anchorage and proliferation, it halts the cell cycle at the G0/G1 change, and it increases the likelihood of cell apoptosis. These are all important in stopping cancer and tumor cells from growing and spreading. The study examined that in certain lung cancers, methylation of some cytosine-guanine sequences, CpG islands, upstream of the PTPRO gene indirectly impedes expression. This is to say that the cancer cells have inhibited the gene that makes their growth and proliferation difficult. The methylation of PTPRO usually happens a large distance upstream of the promoter region and is believed to adjust the chromatin structure in such a way that restricts genomic expression. There are six mRNA variants of human PTPRO with full length and truncated versions. These types were examined under several different conditions to look at methylation and its effects. Mutagenically separated PCR, or MS-PCR, was performed on 42 different lung cancer cell lines along with normal lung cell controls. The analysis showed high levels of methylation in tumor cell CpG islands and little in that of normal lung cells.

The next step for the researchers was to show that it was this methylation that was responsible for restricting the gene expression. Lung cancer cell line A549 cells, with known PTPRO suppression, were exposed to DNA methyltransferases, DAC, and activation was restored after 72 hours. The methylation in the CpG islands was removed and transcription could occur. The researchers were able to see this with the aid of real time PCR, or RT-PCR. This was the support needed to show that it is indeed the methylation that is responsible for the restricted expression, and silencing of PTPRO in the lung cancer cells, specifically the cell line A549.

A549 cells were also transfected with wild type PTPRO and cysteine-serine mutant PTPRO. Colonies of these cells were shown under unrestricted growth with soft-agar assay. The PTPRO wild type cells showed significantly fewer colonies and overall size than both the control and the CS mutant. RT-PCR was done to see the level of expression. The result was shown that over expression of PTPRO inhibits anchorage-independent growth in at least the A549 cells. An MTT assay, which measures metabolic cell rate, was performed over six days using the same A549 cells (wt and cs). The assay demonstrated that those cells with over-expressing PTPRO had less growth and fewer divisions than those in the control. The researcher was able to assume that over expressing PTPRO interferes with the cell cycle and prevents reentry of the cells into S phase. Because of this interference with cell growth, the researchers looked at PTPRO‰Ûªs involvement in apoptosis. The cells were treated with staurosporine, a common apoptosis inducer. After an hour, wild type cells showed degenerating membranes and overall dying cells. The controls and mutant did not show any signs of cell death. This gives support to the idea that over expressing PTPRO aids in apoptosis of the cancer cells in A549 lineages.

It is easy to see that the role of PTPRO has a significant effect in lung cancer cells. This is important for further studies because it may lead to preventative drugs or methods for stopping the spread and growth of cancer.

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Nov. 22, 2010 Presentations

Interdigital webbing retention in bat wings illustrates genetic changes underlying amniote limb diversification

Presented by Mandy Fobar

There has been much research on programmed cell death (PCD)/apoptosis in the interdigital region of mice, chicks, and ducks; but the mechanism and proteins involved in this particular regulation in bats is poorly understood. Scientists compared homologous structures then proteins amongst these different animals including: Bmp (bone morphogenetic protein) which is suggested to incite PCD; Gremlin, a Bmp inhibitor found in ducks which suppresses PCD; and their downstream targets (specifically Msx genes). Also Fgf8 expression was observed in the bat forelimb where little PCD occurred. It was hypothesized then concluded that a combination of Bmp inhibition and Fgf activation are responsible for prevention of interdigital cell death in the forelimb/wing of bats.

First, Bmp expression and Msx gene expression in bat forelimbs and hindlimbs were examined before and at the start of interdigit mesenchyme regression. Prior to regression Bmp2 was expressed relatively evenly throughout all of the hindlimb interdigits, but once regression began Bmp2 was restricted distally (primarily to digits III-V); this is different from other animals which maintain a more even distribution of Bmp2 throughout regression. But Bmp4 shows little interdigit expression in bat forelimbs and hindlimbs, and rather is concentrated near the AER (apical ectodermal ridge) of developing structures similar to mice. Bmp7 is expressed subadjacent to the AER in the most distal interdigit tissues. It was expected since Msx gene expression is downstream of Bmp signaling, that inhibition of Bmp would result in reduction or absence of Msx expression, but these genes were found highly expressed throughout the interdigits in forelimbs and hindlimbs; therefore Msx expr ession is not a strong indicator of potential interdigital cell death.

Next, levels of Gremlin and Fgf8 were examined. Prior regression, Gremlin was expressed most in anterior digits and progressively less in distal digits in the forelimb and also in all interdigital regions of the hindlimb. As regression continues, Gremlin is no longer expressed in the hindlimb but only found flanking the digits and at their tips. Fgf8 (known to block cell death in mice) is expressed in limb mesenchyme, especially at AER, throughout regression. Fgf8 is found at a high concentration in the bat forelimb and at a lower concentration in bat hindlimb. This evidence is indicative that Fgf8 signaling may maintain the interdigital tissue of the bat wing.

Finally scientists tested the role of Fgf and Bmp signaling during interdigital cell death in the bat forelimb by co-inhibiting both; their hypothesis was that apoptosis would be induced and therefore hinder proper formation of the wing. A bead soaked in respective inhibitors was inserted into a developing bat forelimb. Much cell death occurred in the AER, however little cell death occurred in bat forelimb interdigital region, but results were still statistically significantly different from control; this indicates that Fgf and Bmp signaling play some part in the regression of limb mesenchyme during wing development.

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Direct regulation of egl-1 and of programmed cell death by the Hox protein MAB-5 and by CEH-20, a C. elegans homolog of Pbx1

Presented by Alexis Galvez

Hox genes are a group of related genes that specify cell fates along the anterior-posterior axis and segment identity of metazoan organisms during embryonic development. These genes are crucial for the proper placement of embryonic segment structure such as legs, antennae, and eyes. In C. elegans and mammals, mutations in these genes result in abnormal patterns of program cell death. However it is unclear whether these genes solely regulate programmed cell death by transcriptional control of cell death genes or if they indirectly play a role in cell death by cell fate transformations.

This study looked at two of the six Hox genes found in the nematode C. elegans: lin-39 and mab-5. lin -39 is required for the cell survival of the VC neurons found in the midregion of the ventral nerve cord. mab-5 is needed for the programmed cell death of the P11.aaap and P12.aaap cells. Studies on lin-39 and mab-5 null mutants, resulted in abnormal patterns of programmed cell death. The Hox proteins LIN-39 and MAB-5 have also been observed to bind DNA cooperatively with a protein co-factor called CEH-20 during mesoderm differentiation. It has been observed that programmed cell death could be regulated by transcriptional control of egl-1; a gene required for programmed cell fate of somatic cells in C. elegans. The purpose of this study was to examine the mechanism that regulates programmed cell death in the P11 and P12 lineages.

The major results of this study are as follows: 1.) Hox co-factor ceh-20 and hox gene mab-5 regulate programmed cell death in cells P(11, 12).aaap. 2.) In P11.aaap, the CEH-20/MAB-5 complex binds to egl-1 and directly regulates egl-1 transcription. 3.) egl-1 induces programmed cell death in P(11,12).aaap cell. 4.) lin-39 and ceh-20 act upstream of or parallel to egl-1 to prevent cell death of the VC neurons. 5.) egl-1 expression is regulated directly in P11.aaap and indirectly in P12.aaap. Direct regulation occurs because egl-1 contains an evolutionarily conserved genomic sequence that includes a Hox/Pbx site to which the CEH-20/MAB-5 complex binds.

This study showed that the mab-5 Hox gene is essential for programmed cell death in P11 and P12 lineages. Also that the CEH-20/MAB-5 complex directly regulates the expression of egl-1 by binding to a conserved sequence found in the gene. The binding of this complex thus induces the programmed cell death of the P11.aaap cell. It was also observed that mab-5 and ceh-20 indirectly induce programmed cell death in the P12 lineage. A similar mechanism for which lin-39 and ceh-20 regulate VC survival was also proposed. A LIN-39/CEH-20 complex may directly repress transcription of egl-1 to ensure the survival of the VC neurons or these Hox proteins could regulate egl-1 indirectly. It was also discussed that some egl-1 human homologs have been known act during hematopoiesis (the formation and development of blood cells) to prevent hemtopoietic malignancies. Thus it was proposed that regulation of the egl-1 homologs by Hox co-factor/Hox gene complexes could be useful in regulating hematopoiesis by promoting or preventing programmed cell death.

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Loss of Aif Function Causes Cell Death in the Mouse Embryo, but the Temporal Progression of Patterning Is Normal

Presented by Devin Welsh

The gene Aif (Pcdc8) encodes the protein AIF (apoptosis inducing factor), which was first identified as a mitochondrial protein that can induce changes characteristic of apoptosis in isolated nuclei. The function of Aif is an essential mediator of normal apoptosis in early mouse embryogenesis, and the formation of the proamniotic cavity is an apoptosis-dependent event which occurs early during embryogenesis in mice. AIF only promotes cell death after it has been released from the mitochondria.

To test the role of Aif in early embryogenesis they compared wild type embryos with Aif null embryos. These embryos were morphologically similar at E7.75, indicating that embryos that never contain a functional Aif gene can still undergo cavitation. To show that maternal AIF protein produced in the oocyte is not responsible for this cavity formation, multiple cell lines were created from crosses of Aif flox/flox or Aif flox/+ females and -Ac-creTg/Tg males. In Aif null, heterozygous, and wild-type embryonic cell lines the rate of cell proliferation was the same. To see if AIF is necessary for cell death later in embryogenesis they examined Aif null embryos from E8.5 and compared them with wild type embryos and found them to be morphologically indistinguishable. A similar distribution of TUNEL positive cells was seen in wild type and Aif null embryos, showing that normal apoptotic cell death can occur in the absence of AIF.

By E9 morphological differences are observable between Aif null embryos and wild type embryos. In Aif null, the anterior brain and somites show reduced size. The reduced size is due to a reduction of the total cell number of the embryo (Aif null embryos at 20-21 somites contained ~1/3 as many cells as wild type embryos of the same age). This reduced size however only causes a slight delay in patterning of the embryo. Hindlimb buds, which normally develop at 27 somites developed normally in the Aif null embryos even though they were much smaller than wild type embryos at the same stage.

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Timing of the onset of a developmental cell death is controlled by transcriptional induction of the C. elegans ced-3 caspase-encoding gene

Presented by Luis Rodriguez

Programmed Cell Death (PCD) plays an intricate role in organismal development by eliminating unwanted cells. When PCD is disrupted it can lead to various diseases which can include cancer and autoimmune diseases. While a canonical pathway of PCD has been identified in C. elegans, there is little that is known about the timing of the genes in the pathway.

This paper shows how the timing of cell death in C. elegans is controlled by the transcription of ced-3. The study focused on cell death of the tail-spike cells, which undergo apoptosis ten times later than most cells destined to die in C. elegans. This late cell death is important because the tail-spike cell shows differentiated features before dying; similar to differentiating features of cells that die in the development of vertebrates.

The researchers use mutant alleles of genes that are known to be part of the canonical cell death pathway to show the cell death pathway in the tail-spike cell is different than the canonical pathway found in a majority of other somatic cells in C. elegans. They determined that egl-1 is only partially required for tail-spike cell death; where the canonical pathway states that egl-1 activation is required for cell death to occur. To test how the timing of cell death was regulated by the transcription of ced-3 they used mutant forms of GFP and observed when and where transcription occurred in the tail-spike cell during embryo development. They found that transcription of ced-3 did not occur in the cells that were programmed for cell death, but is transcribed in their precursors.

The fact that the tail-spike cell has differences to the canonical pathway suggests that differentiated cells, like the tail-spike, follow an alternate cell death pathway. The results also unveil a new role of ced-3 in controlling the timing of cell death during animal development.

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Nov. 12, 2010 Presentations

Fibroblast growth factor blocks Sonic hedgehog signaling in neuronal precursors and tumor cells

Presented by Sarah Jones

The central nervous system is patterned by gradients of secreted signals called morphogens. A few protein families are central in this pattern formation including the hedgehog family and the fibroblast growth factors. Though Sonic hedgehog (Shh) was originally found to pattern the dorsal ventral axis of the spinal cord it has been discovered to also effect the proliferation of neurons in the cerebellum, one specific instance being granule cell precursors (GCPs). Fibroblast growth factors also have an important role in nervous system development and in the cerebellum they have been linked to axon growth and branching. These two co-ocurring signals have been shown to regulate many of the same processes however this paper demonstrates that they do not necessarily work together. This paper concludes that when these two signals are found together in the cerebellum that basic fibroblast growth factor (bFGF) actually inhibits the mitogenic response of GCPs to Shh. As unregulated GCP proliferation is thought to be one of the leading causes of a form of pediatric cancer, the medulloblastoma tumor, the in vivo results of these experiments could be beneficial in cancer research.

To begin with the paper looks at dispelling some research that seemed contradictory. The authors of the paper had previously published reports that bFGF does inhibit the proliferation of GCPs while other published articles have said that bFGF increases mitosis. By sorting cells with GFP+ signal (meaning they were GCPs and completely purified) they were able to distinguish that in the cerebellum bFGF inhibits proliferation of GCPs. They then demonstrate that in culture, Shh causes proliferation of GCPs and that this effect is disrupted by the addition of bFGF in a dose dependent manner.

They then tested the mechanism by which bFGF worked. As fibroblast growth factors function through an RTK pathway, they tested other known RTK activating growth factors. Only bFGF was able to inhibit Shh proliferation, suggesting that only FGFs have this function. They were then able to determine that bFGF inhibited Shh signal transduction through FGF receptors (FGFRs). They also showed that bFGF is functional through the activation of MAPKs which is typical of this group of growth factors. Lastly, the paper looks at the result of bFGF on the GCPs to see what is happening instead of the proliferative response to Shh. Cells that were treated with Shh alone displayed the typical rounded morphology of dividing cells. Cells which were treated with Shh and bFGF displayed a more typical ÒneuronalÓ morphology and began to extend processes. This suggests that bFGF inhibits the Shh mediated proliferation by promoting GCPs to exit the cell cycle and differentiate.

The most important part of this paper was that they were able to demonstrate the above-described effects of bFGF on proliferating GCPs both in vitro and in vivo. This provides hopeful information that perhaps bFGF could be a useful therapy for medulloblastoma tumors, which are thought to arise from uninhibited proliferation of GCPs. Though the complete pathway of FGF interaction in GCPs is not fully understood, this preliminary data does support the idea that Shh signaling may be a target.

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Fetal alcohol exposure impairs hedgehog cholesterol modification and signaling

Presented by Tim Kaatman

This paper was exploring the relationship between varying levels of alcohol exposure and the spectrum of phenotypic effects that it causes in developing zebrafish embryos. The fetal morphogen Sonic Hedgehog has many roles in cell proliferation, differentiation, and embryonic patterning. Shh protein undergoes post-translational modification by a covalent link of cholesterol to the N terminal region. The now mature Shh protein can be transported to the cell membrane for secretion. Once it has been transported, it initiates the following signal transduction pathway: binds to receptor Patched (Ptc), activates signal transducer Smoothened (Smo), which activates Gli transcription factors which regulates the expression of target genes. Shh is expressed in Henson's node, the floor plate of the neural tube, cardiac mesenchyme, gut endoderm, posterior limb buds, and in the notochord. Shh also affects distal tissues that are developing including: the ventral neural tube, the anterior-posterior limb axis, and the ventral somites. The floor plate of the neural tube regulates the development of motor neuron precursor cells. Shh also has a role in neural crest morphogenesis.

Embryos exposed to alcohol have similar phenotypic defects as those embryos that have defects in hedgehog signal transduction. Ethanol reduces cholesterol levels in cells which is crucial to the modification of Shh to result in its expression. Fetal Alcohol Spectrum Defects (FASD) include neurological, craniofacial, cardiac, limb malformations, and growth retardation. However, alcohol teratogenesis is highly variable.

In their experiment they exposed zebrafish embryos with chorions to six different ethanol concentrations: 0, 0.25, 0.5, 1.0, 1.5, and 2.0%. After exposure, embryos were harvested for phenotypic analysis, hedgehog pathway activity, and cholesterol content. Embryos were also exposed at two different stages: 1 to 2 cell stages to 3hpf and 4.25 to 10.25hpf in late blastula/early gastrula stage. Embryos exposed during the first stage had a higher mortality rate than those exposed at the later stage. The survival of the embryos in the later stage depended on alcohol concentration. Embryos were compared to those exposed to Cyclopamine which inhibits Hh signaling and those exposed to AY-9944 which inhibits cholesterol biosynthesis and transportation. All three groups had phenotypic similarities of Fetal Alcohol Spectrum Defects, which suggests that alcohol disrupts the Shh pathway by inhibiting cholesterol modification. Also cholesterol supplementation restores the Hh signaling pathway and prevents the phenotypic defects. The zebrafish defects as a result of ethanol exposure mirror the defects that humans encounter as a result of ethanol exposure.

These alcohol concentrations were used because they can be reached in the blood from a 121 lbs woman drinking one 12 oz. beer. This is suggesting that there is no safe level of alcohol to drink during pregnancy. The zebrafish model demonstrates that cholesterol supplementation could be a future strategy to inhibit FASD.

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The p75 Neurotrophin Receptor Is a Central Regulator of Glioma Invasion

Presented by Andrew Fleming

Gliomas are malignant, highly invasive tumors of the central nervous system (CNS). The distinguishing feature of gliomas is their aggressive migration and extension of tendrils up to several centimeters away from the main tumor mass, making them virtually untreatable by conventional means. These invasive properties are achieved by activating specific cellular programs that enable them to migrate and to degrade extracellular matrix proteins. The regulation of these specific cellular pathways was investigated in hopes of identifying potential therapeutic targets.

Green fluorescent protein (GFP)-expressing migratory human glioma cells were isolated from non-invasive tumor cells. RNA extracted from the invasive and the control cell lines was used to prepare labeled cDNA for microarray analysis. Among the genes whose expression was significantly up regulated in the glioma cells, p75 was selected for further study. P75^NTR is a ligand-activated neurotrophin receptor that binds a wide variety of neurotrophins including BDNF, NT-3, and NGF. Although p75 and its ligands are known to play a role in neuronal growth, survival, and differentiation during development, their participation in tumors has not been previously described.

The noninvasive and highly invasive cells were treated with NGF (Neuron Growth Factor). In the invasive cell line, a striking increase in the ability to invade through gelatin, even at low concentrations, was observed. However, it was noted that the invasive cells also had increased migration in the absence of NGF, suggesting that glioma cells expressing the P75^NTR receptor activate an autocrine loop by producing and secreting neurotrophins.

To test whether elevated expression of P75^NTR is necessary for migration and invasion, RNA interference (RNAi) was used to down regulate P75^NTR in cells endogenously expressing the receptor. This cell line was injected with P75^NTR-specific small interfering RNA (siRNA), resulting in reduced ability to migrate, and rendering the cells unresponsive to NGF. Conversely, cDNA from human P75^NTR was transfected into a cell line that expressed no endogenous P75^NTR. Ectopic expression of P75^NTR in these cells was sufficient to cause an increase in migration and invasion.

To reproduce these results in-vivo, the brains of immuno-compromised mice were implanted with either the P75^NTR-expressing glioma cell line or the normal tumor cell line. After an incubation period, the brains were stained for P75^NTR expression. Implantation of the control cell line led to tumors with well-defined borders. Implantation of the P75^NTR-expressing cell line resulted in tumors with highly invasive edges and migrating glioma cells. In-vivo experiments using another, genetically distinct cell line yielded the same results. Mutant cell lines altered at the ligand-binding domain of the P75^NTR gene were constructed. In wild type glioma cells, expression of P75^NTR caused a shift in localization of BDNF (a p75 ligand) to the interior of the cell. In P75^NTR mutants, BDNF was only observed on the plate medium. Implantation of P75^NTR mutants into immuno-compromised mice brains led to tumors with well-defined borders, indicating neurotrophin binding is required for glioma invasion. Invasive glioma cells also display drastic changes in cytoskeletal structure, including numerous filamentous protrusions. Expression of P75^NTR alone was demonstrated to induce these structural rearrangements. In conclusion, neurotrophin-dependent P75^NTR was shown to be an important regulator of glioma invasion and migration.

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Sonic Hedgehog Guides Axons through a Noncanonical, Src-Family-Kinase-Dependent Signaling Pathway

Presented by Jennifer Shaw

Sonic hedgehog plays an important role in development. Two of its most important roles concern cell fate specification and axon guidance, which are two distinct cellular processes. Specifically, this paper describes the role of Sonic hedgehog in axon guidance. The experiments performed attempt to unravel the mechanism by which this occurs. To develop hypotheses for this mechanism, mechanisms of other axon guidance cues were examined. For example, netrin and semaphorin guide axons by inducing local changes at the growth cone, and neither the cell body nor transcriptional activity is required for axon turning. Experiments were designed using these facts to see if Sonic hedgehog functions in a similar manner to guide commissural axons.

Three major experiments were performed. The first was an experiment to determine if Shh does in fact cause axon turning. As a control, commissural neurons were exposed to a gradient of bovine serum albumin, the ÒvehicleÓ of Shh, in a Dunn chamber. These neurons continued to grow without any change of direction. When exposed to a gradient of Shh, however, the commissural neurons turned up the gradient. There were no significant differences in the amount of growth seen using the control and Shh. This supports previous experiments that Shh is not a growth factor for this type of neuron. Inhibiting Smoothened, a transmembrane protein that activates the Shh pathway, prevented axon turning up the Shh gradient. This first experiment shows that Shh promotes axon turning and that Smoothened is required for this to occur.

The second major experiment was to determine if transcriptional activity is required for Shh-mediated axon guidance. The time it took for commissural neurons to turn up the gradient was measured. The median time was about eight minutes, and there was no time gap between the start of axon turning and the start of axon growth. This suggests that Shh-mediated axon guidance could occur without transcriptional activity simply because transcription takes time. To directly test this hypothesis, transcription was blocked in commissural neurons using two different drugs. Adding these inhibitors did not prevent axon turning up the Shh gradient. Therefore, Shh-mediated axon guidance does not require transcriptional activity. The targets of the Shh pathway are the Gli family of the proteins (transcription factors). Inhibiting the transcription of these proteins also did not prevent axon turning up the Shh gradient. This further suggests hat Shh must mediate axon guidance in a non-canonical fashion.

The third major experiment was to determine if SFKs play an instructive role in Shh-mediated axon guidance. SFKs, or Src family kinases, are found on commissural neurons. Commissural neurons were exposed to an Shh gradient in a Dunn chamber and then immunostained for SFKs. Higher SFK levels were found on the parts of the growth cone facing higher levels of Shh, suggesting that Shh increases the presence of SFKs. To determine whether SFK activity is required for axon guidance, SFK activity was inhibited in commissural neurons and exposed to a Shh gradient. These neurons did not show axon turning. This suggests that SFK activity is required for Shh-mediated axon guidance. In conclusion, Shh mediates axon guidance in a non-canonical, transcriptionally-independent manner.

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