"Read More About It" Pages - Biology 376 - Fall 2011

Dec. 7, 2011 Presentations

pRB Inactivation in Mammary Cells Reveals Common Mechanisms for Tumor Initiation and Progression in Divergent Epithelia

Retinoblastoma (pRB) and related proteins play a significant role in regulating eukaryotic cell cycle progression and apoptosis. Lack of pRB function is known to lead to increased profileration, apoptosis, and cancer predisposition. In addition, P53 is also commonly mutated in cancers. This paper discusses the effects of both pRB and p53 inactivation on the predisposition and progression of breast cancer in mice.

The researchers created transgenic mice by inserting a truncation mutant, T121, of the SV40 T antigen, which inactivated all RB family proteins. They found that the inactivation of pRB and related proteins led to increased proliferation and apoptosis. They also found that it took an average time of ten months for pRB-deficient mice to develop tumors in the line of mice with the highest amount of T121, and all had tumors by sixteen months.

To assess the role of P53 in cancer development and progression, the researchers mated P53 null mice and T121/pRB deficient mice. They found that lack of P53 corresponded with a lower percentage of apoptotic cells, revealing that a large majority of apoptosis is P53-dependent. In addition, tumor development in P53 heterozygous mice was shown to be detected earlier and have a faster growth rate in comparison to P53 wild type mice. Therefore, the onset and growth of tumors is accelerated by P53 reduction in breast cancer.

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Cellular senescence is an important mechanism of tumor regression upon c-Myc inactivation

The c-Myc (MYC) proto-oncogene is a transcription factor that has an important role in the regulation of many cellular programs. Among these programs are proliferation, differentiation, and apoptosis. Consequently overexpression or a mutation of MYC is commonly associated with tumorigenesis, unlimited cell proliferation, and no apoptosis. The cancer gene MYC is among the most commonly overexpressed oncogenes in human cancers.

To further understand MYC as an oncogene, an experiment was preformed on transgenic mice that overexpress MYC by using a regulatory system called Tet-off. Once cancer formed in the mice, MYC was then inactivated in order to understand MYC's role in tumorigenesis. It was shown that MYC induced tumor regression by a cellular mechanism called senescence. Senescence is a growth arrest program that prevents unlimited cell proliferation.

There are multiple factors observed in this experiment that are directly related to cellular senescence when the MYC oncogene is inactivated. It is shown that there is an increase in cellular senescence markers, changes in chromatin structure (euchromatin becomes heterochromatin), and MYC inactivation is p53 dependent. Also, the signaling pathways associated with MYC inactivation did not have a direct effect on senescence. When these pathways (ATM/ATR and MAPK) were inhibited, senescence was still able to occur in the MYC oncogene.

Overall, this article shows that by repressing the MYC oncogene and cellular senescence program occurs, the transformation process of a tumor can be suppressed.

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WNT5A Exhibits Tumor-Suppressive Activity through Antagonizing Wnt/Beta-Catenin Signaling, and Is Frequently Methylated in Colorectal Cancer

WNT5A is a noncanonical, nontransforming Wnt protein located at 3p14 which is a commonly deleted tumor suppressor locus in multiple tumors. On one hand, it has a role in lung, some breast, stomach, and prostate cancers as a proto-oncogene, but in cancers regarding the hemotopoietic and uroepithelial tissues, the brain, breast, and thyroid, there are data suggesting that it inhibits tumor cell proliferation.

Abnormal promoter methylation causes tumor suppressor genes to become inactive. In all tumor types tumor suppressor genes such as WNT5A are silenced by aberrant CpG methylation. When they demethylated WNT5A, they found that it promotes β-catenin degradation, thus antagonizing canonical Wnt signaling. In that way, it inhibits the clonogenicity of CRC cells, as suggested by colony formation suppression findings in their experiment.

Because of silencing in CRC cells via tumor-specific methylation, WNT5A can be used as potential prognostic biomarker.

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Telomerase modulates Wnt signalling by association with target gene chromatin

Telomerase has been shown to have a role in cell proliferation, cell survival, and chromatin regulation activities, but surprisingly this role has been demonstrated to be independent of its telomere-lengthening activities. In their research, Park et al. further investigated the role of telomerase - focusing on telomerase reverse transcriptase (TERT) - to better understand the mechanism by which it functions.

The researchers discovered that TERT plays a role in the Wnt signaling pathway by serving as a cofactor to beta-catenin. They used immunoprecipitation tests and found that TERT interacts with the protein BRG1, which is known to change chromatin structure. As a result, they tested the presence of TERT in Wnt target gene promoters, and found expression of TERT in Wnt target gene promoters such as Axin-2 and Myc.

The researchers also found that TERT has an effect in Xenopus anterior-posterior axis formation. When they injected beta-catenin and TERT into the posterior cells of developing Xenopus embryos, they found that an entirely separate A-P axis developed from the same embryo, such that it had "two heads." TERT is significant in the Wnt pathway in that it acts as a transcription cofactor with beta-catenin for Wnt target genes.

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Nov. 30, 2011 Presentations

WNT/TCF Signaling through LEF1 and HOXB9 Mediates Lung Adenocarcinoma Metastasis

Lung cancer metastasis usually occurs within months of diagnosis, much more rapidly than other forms of cancer and by a previously unknown mechanism. The brain and bone are sites that usually show a rapid onset of tumor growth once this differentiation occurs and metastasis begins. This paper's hypothesis is that lung adenocarcinomas with high competence for metastasis may be distinguished by TCF4 activation through a canonical Wnt pathway.

The Wnt pathway under investigation for this study effects the transcription of LEF1 and HOXB9 genes. The paper uses several overlapping studies in order to connect this specific pathway with lung adenocarcinoma metastasis to the brain and bone tissues. New cell lines were created for the study using gene sets prone to the select mutations for this type of metastasis. The original cells for the study were isolated from the lymph nodes of lung cancer patients.

Because the cells were originally isolated from lymph nodes, this suggests the hyperactive Wnt pathway has already appeared in cells before they leave the lungs. The results of this study show activation LEF1 and HOXB9 transcription factors are responsible for the ability of lung adenocarcinoma to metastasize to other organs especially the lungs and bone.

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β-Catenin induces immortalization of melanocytes by suppressing p16INK4a expression and cooperates with N-Ras in melanoma development.

Melanoma is a form of cancer on the skin. It is caused by mutations in skin epithelial melanocyte cells. Melanocytes produce skin pigment, melanin, but mutations can cause tumor growth and cancer. Primary causes of these mutations are UV radiation from the sun. Melanoma can occur anywhere on the body that can be exposed to UV radiation.

This article focuses on β-catenin's effects upon p16INK4a and its potential to cause melanoma when associated with N-Ras. β-catenin is part of the Wnt signaling pathway and is an essential part for transcription of genes. It was found to suppress p16INK4a.

p16INK4a is a protein that is an indicator of cell age, senescence. It is also a tumor suppressor. The more protein present means the older the cell is and it needs to die. Suppression of p16INK4a expression would trick cells into “thinking” that they are young forever. The 'immortalization' of cells leads to cancer; they just don't die. Immortalization is quite different from proliferation of cells during tumor growth. It is like children getting on the school bus and not leaving, whereas proliferation can be represented by excessive amount of children getting on the bus because of a rainy day. In both cases, the bus is packed to the max but for different reasons. In this article, it was found that immortalization is a result of β-catenin suppressing p16INK4a expression. Delmas et al find that the suppression of p16INK4a is not the only cause of melanoma, but needs to be present in mutants for N-Ras.

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Aging and cancer resistance in lymphoid progenitors are linked processes conferred by p16 Ink4a and Arf

Lymphoid progenitor cells create new white blood cells and can be found in the bone marrow. They have been found to exhibit growth defects during the aging process and are linked to tumor suppressor proteins, p16ink4a and Arf. These proteins regulate the cell cycle, more specifically cell senescence. Mutations in the expression of p16ink4a and Arf contribute to the reduced growth and survival of lymphoid progenitor cells which can lead to the formation of malignant tumors and cancers.

Lymphoid lineage is linked to p16ink4a and Arf in it is sensitivity to the effects of aging. The common lymphoid progenitors, pre-pro-B, pro-B, and pre-B cells are found to significantly decline in frequency and quantity when exposed to deficits in proliferation and survival of cells. In an experiment conducted by Signer et al., aged lymphoid progenitors were found to have reduced leukemonegic potential. When pro-B and pre-B cells were isolated from the bone marrow of young mice and transformed using a subset of pre-B lympohoblastic leukemia in vitro, the cells generated lymphocytes in excessive quantities (lymphoproliferative disease) which heave lead to leukemia. However, when B-cell lymphoid progenitors from older mice were used, findings showed minimal excess proliferation and did not generate leukemia.

A key marker of aging in mice and human tissues is the increased expression of the tumor suppressor proteins, p16ink4a and Arf. This increase in expression has been found to protect against cancer by inducing senescence as a response to the buildup of oncogenic stressors such as DNA damage, oxidative species, and telomere mutations all accumulated over aging. Experiments in the removal of p16ink4a and Arf have been found to increase expression of leukemia in adults.

This experiment highlights the possible connections between aging, cancer, and leukemia in genetic mechanisms attributed to a decline in the production of white blood cells. This processes have linked aging functionality in cells as a promoter of resistance against leukemogenesis.

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Targeted depletion of Polo-like kinase (Plk) 1 via lentiviral shRNA or a small-molecule inhibitor causes mitotic catastrophe and induction of apoptosis in human melanoma cells

Melanoma is extremely aggressive and is one of the more lethal forms of skin cancer. Unfortunately, the majority of the available therapies for dealing with metastatic skin cancers have about a 25% success rate, and none of these are able to prevent the growth of this cancer on other areas of the body. In order to have normal skin cell turn over, there must be Òerror-free chromosomal segregation during cell divisionÓ. Without this, diseases, including cancer, can occur.

There are two major components of cell division that have to be executed perfectly in order to have normal cell growth. First, the chromosomes have to segregate equally and second, cytokenesis has to occur properly. In order for this to work properly, one key player has to be functioning properly as well. Polo-like kinase 1 (Plk1) belongs to a highly conserved group of serine/theronine protein kinases. These Plks are found to be homologous across many species in the animal kingdom, ranging from yeast to humans. There are 4 kinds of Plks in mammals and Plk1 is by far the most widely studied. Plk1 is controlled by mRNA accumulation throughout various stages of the cell cycle. It is found that there are high concentrations of mRNAs at the G2-M transition and protein levels along with kinase activity mimics these increases as well. The role of Plk1 includes: Òactivation of the Cdk1/cyclin B1 cascade, centrosome maturation, bipolar spindle formation, the regulation of Emi1 degradation at mitotic exit and cytokinesis. Plk1 is also able to destabilize and reduce p53 activity through direct binding and phosphorylation of p53Ó. It has been suggested that Plk1 is an oncogene from two major findings. First, the dysregulation of Plk1 causes abnormal centromeres to form and over-expression of Plk1 causes this phenotype, which results in tumor cells. Second, it was also found that the inhibition of Plk1 shows a decrease in cancerous skin cells. However, the actual function of Plk1 is poorly understood, especially in the case of melanoma. This paper looked at what that function could possibly be in hopes of possibly building a base to begin research into possible novel techniques of melanoma treatment.

The first test compared 40 normal human skin samples and 37 malignant melanoma samples ranging in severity of invasiveness. It was f ound that melanoma samples had high concentrations of Plk1 compared to the normal skin samples and the severity of the invasiveness directly correlated to the concentration of Plk1 in the cells. This was determined by simple cell staining techniques. Next, they looked into whether or not the mRNA expression as well as the expression of associated proteins reflects this increase. It was found (by use of real-time PCR) that the mRNA and the protein concentrations are significantly higher in tumor cells compared to normal skin cells. After, they inhibited Plk1 to see if there were any effects on cell turn over. Knocking out the Plk1 gene resulted in a dramatic reduction in tumor cells, suggesting that these high concentrations are necessary for tumor cells to continue to exist. Finally, they looked at what would happen if the cell cycle were to be interrupted. By inhibiting Plk1 in the G2-M transition, tumor cells in this phase quickly accumulated. Upon analyzing these cells, it was found that the typical Òpolo-phenotypeÓ was present in most cells. Other errors were found as well, all of which resulted in improper cell division and tumor cell development. This however, kept the cells in this phase and potentially could tag the cell for apoptosis, which was found to be true through a flow cytometric evaluation of these tumor cells.

Overall, it was found that over-expression of Plk1 can lead to melanoma skin cells and that controlling the concentration of Plk1 in cells results in removal of melanoma cells by apoptosis. This suggests that Plk1 can potentially major role in future treatment of skin cancers. However, more studies need to be conducted in order to find out how to go about utilizing this information for creating novel methods of treatment.

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Nov. 21, 2011 Presentations

Ethanol causes the redistribution of L1 cell adhesion molecule in lipid rafts

L1 is a cell adhesion molecule belonging to the Ig superfamily. Previously, mutations in L1 have closely resembled those effects of fetal alcohol syndrome, a detrimental defect caused by ethanol consumption during pregnancy. The L1 molecule plays an integral role in neurile outgrowth by binding to neurons expressing L1 on the exteriors of the cells. Lipid rafts, part of the dynamic plasma membrane, have been shown to closely associate with L1 molecules. Although how this is regulated remains unknown, the disruption of lipid rafts has been proven to reduce neurite outgrowth on L1. The disruption of this relationship may be the key in identifying ethanol's toxicity.

Tang et al. tested their hypothesis using CAMs from goats, and N-cadherin natibodies from mice. Isolations and preparation of CGN cells are from rats and are treated with ethanol one hour before harvesting the cells. A gradient is then run to separate lip raft containing fractions from those fractions lacking lipid rafts. Cells are plated and fixed using several controls. Two hours post-plating ethanol is added to some of the cultures. At 12 hours the cells are fixed and a non-biased researched chooses the neurite for measurement

In order to isolate the importance of disturbance on the L1 CAM specifically ethanol was shown to have no effect on N-cadherin distribution. N-cadherin existed in lipid rafts in both control cells and affected cells, showing no shift. In lipid rafts exposed to ethanol, the amount of beta-tubululin, microtubules that mediate lipid rafts, decreased. Redistribution of L1 into lipid rafts, causing FAS effects, occurred at relatively low concentrations of ethanol. When measuring neurite lengths, those CGN cells exposed to low concentrations of ethanol had reduced neurite lengths by 70% compared to the controls.

This experiment reveals the dependence of L1 neurite growth on lipid raft organization. Disruption of the L1 trafficking in and out of these lipid rafts is caused by the exposure to ethanol. In this way, L1 can no longer communicate or associate closely with the lipid rafts. Due to either the increase in L1 trafficking to the lipid raft, or a decrease in signaling outward, an increase in L1 within the lipid raft is seen. The potential connection to FAS exists, although stronger implications are needed for therapeutic outcomes.

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A BMP-Shh negative-feedback loop restricts Shh expression during limb development

The sonic hedgehog (Shh) gene is vital for limb bud formation in animals. Its expression is tightly controlled, particularly in the zone of polarizing activity (ZPA). Many factors are responsible for this level of control in the gene's expression, and not all is known about what restricts and maintains it. It is known that the FGF and Wnt signaling pathways play a large role, and it is thought that the expression of BMP influences these pathways.

Felix Bastila et al decided to determine how and in what way BMP influences Shh expression in the limb buds of mice and chicks. They did this by loading an acrylic bead with BMP2 and inserting it into the posterior mesoderm of the limb bud of each embryo. The control groups had beads soaked in the vehicle solution implanted. They then stained and observed the limb bud so that they could visually detect the patterns of Shh expression. The BMP-loaded beads restricted the expression of Shh in the limb bud for up to 22 hours after insertion. Conversely, the control group showed no such effect.

In order to determine how BMP restricts Shh expression, Bastilda et al ran another experiment where they implanted beads soaked in FGF8, a key component of the FGF signaling pathway, into the mesoderm of the limb bud before implanting the BMP beads. The FGF8 beads expanded the range of Shh expression, but when BMP was brought in the pattern of expression shrank drastically. This shows that BMP negatively affects the FGF signaling pathway. The Wnt signaling pathway was similarly tested with a bead soaked in Wnt7, but the addition of a BMP bead did not significantly affect it.

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Apoptosis Initiated When BH3 Ligands Engage Multiple Bcl-2 Homologs, Not Bax or Bak

Mitochondria are very important in the facilitation of apoptosis in mammals, as they release a number of pro-apoptotic proteins, like Apoptosis Inducing Factor (AIF) and cytochrome c, through the Permeability Transition pore. This pore is activated by Bcl-2 homology domain 3 proteins, or BH3-only proteins, and is crucial to successful regulation of apoptosis in cells.

These BH3-only proteins, which includes the proteins Bim, Bid, Puma, Bad, and Noxa, all function to sense cellular damage. When this damage is sensed, it suppresses the expression of the Bax and Bak proteins, the absence of which abolishes the apoptotic response. Bax and Bak together promote the permeability of the outer mitochondrial membrane, which allows cytochrome c to activate the caspases the regulate cellular demolition. However, a very important question remains: do these BH3-only proteins activate the apoptotic process by binding directly to Bax and Bak, or do they they act indirectly by binding to Bcl-2 like relatives like Bcl-Xl or Mcl-1. This paper offers evidence that these BH3-only proteins engage these Bcl-2 like relatives directly.

Testing of different cell lysates and combinations of the different proteins shows strong evidence of the indirect activation model, which states that BH3-only proteins can initiate apoptosis without binding Bax or Bak. This emphasizes the central role of mammalian BCL-2 proteins as pro-survival factors, as BH3-only proteins trigger apoptosis by neutralizing these proteins and allowing Bak and Bax to proceed. This is a significant step forward in our understanding and thus potential to regulate apoptosis.

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Ethanol exposure during neurogenesis induces persistent effects on neural maturation: evidence from an ex vivo model of fetal cerebral cortical neuroepithelial progenitor maturation

Alcohol is a well-known teratogen and children with Fetal Alcohol Syndrome exhibit defects associated with neuronal migration. Much research has been done studying the effects of ethanol in the third trimester of fetal development because of the severe defects resulting from exposure, often resulting in death of many parts of the brain. As the second trimester is an important period in neuronal proliferation and differentiation, Camarillo and Miranda focused on the effects of ethanol exposure to mouse neuroepithelial cells during this time. Specifically, their goal was to determine both the immediate and persistent effects of alcohol on neuronal migration.

Three main aspects of neuronal development were studied: cell migration gene expression, neuronal migration, and neurite length. As in vivo alcohol exposure often results from either episodic binge drinking or chronic alcoholism, both levels of alcohol (120 mg/dl and 320 mg/dl, respectively) were used during testing. Results showed that alcohol had immediate effects on cell migration genes, and both alcohol levels caused neurons to migrate significantly farther after proliferation. Alcohol also caused significant increases in primary neurite length, but less secondary neurite branches.

Their conclusion was that the data from this experiment support the observations of displaced neurons, which characterize the brains of infants diagnosed with fetal alcohol syndrome.

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