240314 Flashcards
Acute myeloid leukemia (AML) is an aggressive hematopoietic malignancy for which there is
an unmet need for novel treatment strategies. There is evidence that the growth arrest and DNA
damage-inducible gene gamma (GADD45g) is a novel tumor suppressor in AML. This finding
may have important clinical implications for cancer therapy. In the laboratory you work, you
have access to different AML cell lines, bone marrow specimens collected from patients with
newly diagnosed AML, human cord blood obtained from healthy volunteers, and NOD/SCID
mice.
A. What type of preliminary experimental evidence would support your initial hypothesis
that the gene GADD45g is involved in AML? Provide at least three examples of
experimental evidence and briefly describe the methodological procedure.
Preliminary Experimental Evidence for the Role of GADD45g in AML
1. Gene Expression Analysis in AML Cell Lines and Patient Samples
Procedure:
Collect RNA from AML cell lines, bone marrow specimens from AML patients, and healthy controls (e.g., cord blood from volunteers).
Perform quantitative PCR (qPCR) or RNA sequencing to measure the mRNA expression levels of GADD45g.
Normalize gene expression to a housekeeping gene (e.g., GAPDH) and compare the levels across groups.
Validate results with immunohistochemistry (IHC) or Western blotting to assess protein levels of GADD45g in the same samples.
Expected Evidence:
Reduced GADD45g expression in AML patient samples and cell lines compared to healthy controls would suggest that it acts as a tumor suppressor.
Correlate low GADD45g levels with clinical outcomes, such as higher disease aggressiveness or poor prognosis.
2. Functional Studies Using GADD45g Knockdown or Overexpression
Procedure:
For knockdown: Transfect healthy hematopoietic cells or AML cell lines with siRNA/shRNA targeting GADD45g.
For overexpression: Transfect AML cell lines with a plasmid or viral vector encoding GADD45g. Include controls with scrambled siRNA/shRNA or empty vectors.
Assess changes in cell proliferation, apoptosis, and cell cycle progression using:
Cell viability assays (e.g., MTT, WST-1, or CellTiter-Glo).
Flow cytometry to analyze apoptosis (Annexin V/PI staining) and cell cycle distribution.
Western blotting to evaluate downstream targets of GADD45g, such as DNA damage markers (p53, γH2AX).
Expected Evidence:
Knockdown of GADD45g in healthy hematopoietic cells should enhance proliferation and survival, supporting its role in growth arrest and DNA damage response.
Overexpression of GADD45g in AML cell lines should suppress proliferation, induce apoptosis, or increase sensitivity to chemotherapeutic agents.
3. In Vivo Tumor Suppression Studies in NOD/SCID Mice
Procedure:
Establish two groups of AML cells: one with GADD45g overexpression and one with GADD45g knockdown.
Inject these cells into immunocompromised NOD/SCID mice to create xenograft models of AML.
Monitor leukemia progression by:
Measuring tumor burden using bioluminescent imaging or flow cytometry of human CD45+ cells in the mouse blood, bone marrow, or spleen.
Assessing overall survival of the mice.
Performing histological analysis of bone marrow and spleen to confirm leukemia progression.
Expected Evidence:
Mice injected with GADD45g-overexpressing AML cells should show reduced tumor burden, slower disease progression, and improved survival compared to controls.
Conversely, mice injected with GADD45g-knockdown cells should exhibit accelerated leukemia progression.
Acute myeloid leukemia (AML) is an aggressive hematopoietic malignancy for which there is
an unmet need for novel treatment strategies. There is evidence that the growth arrest and DNA
damage-inducible gene gamma (GADD45g) is a novel tumor suppressor in AML. This finding
may have important clinical implications for cancer therapy. In the laboratory you work, you
have access to different AML cell lines, bone marrow specimens collected from patients with
newly diagnosed AML, human cord blood obtained from healthy volunteers, and NOD/SCID
mice.
. What does the Figure 1 present regarding the GADD45g expression? What is the name
of such plot and what is it used for in cancer research? What is the probability
(approximately) that patients with GADD45g-high will survive beyond 30 months?
What is the probability (approximately) that patients with GADD45g-low will survive
beyond 20 months?
The figure shows a Kaplan-Meier survival curve that shows the cumulative survival probabilities. One axis shows the overall surrvival rate and the other one shows the time for example in months. The slope usually goes down with time and there can be different slopes with different properties of the patients such as high or low GADD45g leading to different surrvival probability rates.
Approximately 60% of patients with high GADD45g expression survive beyond 30 months.
Approximately 37% of patients with low GADD45g expression survive beyond 20 months.
How does understanding of defective DNA repair processes in tumor cells make possible the
development of new anti-cancer therapeutic startegies? An example of the defective DNA
repair process and its targeting should be considered in your answer.
There are different DNA repair pathways such as homologous recombination, mismatch repair genes, etc. Since this causes tumor cells to rely on other DNA repair mechanisms by inhibiting those repair mechanisms we can more selectively target the cancer cells while help spare the healthy cells that still has fuinctional homologous reapir or mismatch repair mechanisms.
For example with BRCA1/2 there is PARP inhibitors that block gthe base excision repair pathway that reapir single strand breaks triggering apoptosis and cell cycle arrest.
Figure 2 presents a model of carcinogenesis. Describe the presented model of carcinogenesis
and use it to illustrate the difference between the hereditary and sporadic form of cancer
The Knudson’s Two-Hit Model: states that a person born with a herediatary mutation one has one healthy allele that can compensate and if they then aquire a second mutation they develop tumorigenesis while non hereditary cancers needs to develop to somatic mutations for this to develop which makes it less likely to occur.
Figure 3 presents a chemical modification of DNA. Describe the mechanism of this process,
subcellular occurrence as well as role of this process in cancer diagnostics.
DNA methylation adds a methyl group to the cytosince base of the DNA. DNMT catalyze this process and when promoter regions of tumor supressor genes are methylated they are silences. Regular genes that are activated by mutations or epigenetic changes such as losing their methylation at the promoter regions can become proto-oncogens and promote cancer.
As a diagnostic tool we can perform sequencing to find proto-oncogenes such as methylation of the BRCA1 gene, liquid biopsy to check methylation markers, and for theraputic targeting so for exampling finding supressed tumor supressor genes or prot-oncogenes and adjusting treatments based on their sensitivity.
What are the molecular mechanisms that lead to the conversion of a proto-oncogene into an
active oncogene? Give example of three known oncogenes and describe their oncogenic mode
of action.
point mutations can cause proto-oncogenes to become hyperactive by for example changing the amino acid sequence. Gene emplification from errors in DNA replication causing multiple copies to go out in the cell leading to an excessive protein. Chromosomal translocation which can activate genes by bringing them closer to an active promoter. A retrovirus can insert itself near a proto-oncogene that becomes upregulated. This can all lead to overexpression and drive cell proliferation, surrvival etc forward.
RAS genes encode for a family of small GTPase proteins (mainly HRAS, KRAS, and NRAS) that regulates cell growth, differentiation, and survival. These proteins act as molecular switches, cycling between an active GTP-bound state and an inactive GDP-bound state. When mutated, they can drive cancer by perpetuating signals that lead to excessive cell division and survival. Point mutations can make them always on. Role in Tumourigenesis: Constitutively active RAS promotes cell proliferation and survival through the MAPK and PI3K-AKT signaling pathways, leading to uncontrolled cell growth.
MYC gene encodes a family of transcription factors (primarily c-Myc, N-Myc, and L-Myc) that regulates various cellular processes, including cell growth, proliferation, differentiation, and apoptosis. MYC is one of the most commonly dysregulated genes in human cancers. Overexpression or amplification of MYC is found in many cancers, including breast cancer, lung cancer, leukemia, lymphoma, and neuroblastoma. This dysregulation typically occurs through mechanisms like gene amplification, chromosomal translocations, or altered signaling pathways that lead to increased MYC protein levels.
The HER2 gene encodes a protein that is part of the EGFR family. HER2 is involved in regulating cell growth, survival, and differentiation. Under normal conditions, HER2 helps control cell division and tissue growth.
In some cancers, particularly breast cancer, the HER2 gene can be amplified or overexpressed, leading to an excessive amount of HER2 protein on the surface of cells. This overexpression causes abnormal signalling, promoting uncontrolled cell growth and contributing to the development and progression of cancer.
A. What is the major role of cancer stem cells in cancer development?
Cancer stem cells have stem cell like proprties such as self renewal and ability to differentiate. They can intiate tumor growth by self renewing, cause tumor heterogenity with their differination, they are often more resistant to therapy due to enhanced DNA repair, drug efflux etc, they can spread to distant sites facilitating metastasis, and can even after therapy remian dormant to then reactivate cuasing cancer relapse.
B. Name and describe two experimental approaches for the study of human cancer stem
cells.
- Sphere Formation Assay (In Vitro Functional Assay)
Description:
CSCs have the unique ability to survive and grow under non-adherent, serum-free culture conditions that inhibit the growth of non-stem cancer cells. The sphere formation assay leverages this property to assess the presence and functionality of CSCs.
Procedure:
Plate single-cell suspensions of tumor cells in a low-adherence culture plate with a defined serum-free medium supplemented with growth factors such as EGF (epidermal growth factor) and bFGF (basic fibroblast growth factor).
Incubate the cells for several days and monitor the formation of free-floating, spherical clusters called tumor spheres.
Quantify the number and size of spheres formed, as they reflect the self-renewal and proliferative capacity of CSCs.
Applications:
Identifies and enriches CSC populations.
Evaluates the impact of drugs or genetic manipulation on CSC self-renewal.
Limitations:
Sphere formation is not exclusive to CSCs; non-CSCs can sometimes form spheres.
Culture conditions may not fully recapitulate the tumor microenvironment. - Xenograft Assay (In Vivo Functional Assay)
Description:
This approach evaluates the tumorigenic potential of CSCs by transplanting them into immunocompromised mice and assessing their ability to initiate and sustain tumor growth.
Procedure:
Isolate CSCs and non-CSCs from a tumor sample using markers associated with CSCs (e.g., CD133, CD44, ALDH). This is often done via flow cytometry or magnetic-activated cell sorting (MACS).
Inject the isolated cells into immunodeficient mice (e.g., NOD/SCID or NSG mice) subcutaneously or orthotopically into the tissue of origin.
Monitor tumor formation, growth rate, and histological characteristics over time.
Compare the tumorigenic capacity of CSCs versus non-CSCs.
Applications:
Confirms the tumorigenic potential of CSCs.
Models the role of CSCs in tumor initiation and progression.
Evaluates CSC-targeting therapies in a physiologically relevant environment.
Limitations:
Requires specialized facilities and ethical approval.
Immunocompromised mice do not fully mimic human immune-tumor interactions.
C. Describe three mechanisms mediating the resistance of cancer stem cells to anti-cancer
therapy.
The cancer stem cells have enhanced dna repair proprties so they can surrvive radiation and chemotherapy better, overexpression of certain genes causes drug efflux vua ABC transporters, and due to quescence and low proliferation rate which protects them against chemotherapy that targets rapidly dividing cells.
The apoptosis mechanism is often altered in cancer cells. Give two examples of regulatory
proteins that are often altered in tumours and describe how they affect the apoptosis
mechanism.
p53 is an important tumor supressor gene that can be mutated in cancer. It activates pro-apoptotic genes such as Bax, PUMA and NOXA in response to cellular stress or damage. Mutation of this leads to continous cell proliferation and avoidance of apoptsosis despite DNA damage.
Bcl-2 is an anti-apoptopic protein that activates the intrinsic pathway by preventing the release of cytochrome c from the mitochondria. It binds and inhibits pro-apoptopic proteins such as Bax and Bak and blocking these blocks the apoptosome formation and caspase activation. The overexpression of this leads to cell surrviging hypoxia and DNA damage.
What is a Vogelstein model and what can we learn from this model?
The vogelstein model shows the accumilation of mutations that is necessary to drive colerectal canciorogenesis forward.
It shows how normal epithelium develop an APC mutation and get genomic instability from this that causes early adenoma and then K-ras driving it into intermediate adenoma, p53 leading it to late adenoma to carcinoma etc.
Describe the two major mechanisms that tumour cells employ to maintain their telomeres.
Telomerase activation which is an enzyme that catalyzes the extension of telomers and has a subunit that serves as a template for the RNA component for the telomerase repeats. In a lot of cancers this is upregulated often through overexpression of the TERT gene which promoter often gets mutated to overexpress in tumors. This allows the cells to dividide indefiently.
Alternative lenghening of telomers (ALT) is telomerase independent and relies on homologous recombination to maintain telomere lenght. This involves exchange of telomeric sequences between sister chromatids or other chromosomes and incorperation of telomeric DNA synthesized using the DNA template from another telomere. Can also be upregulated in cancer but less common but leads to heterogenous telomers.
Tumours are classified into broad classes based on their cell of origin. Describe 4 of these broad
classes of tumours, cell from which they arise and list two examples of specific tumour types
that belong to each tumour class.
Epithelial tumors also called carcinomas come from epithelial cells so surfaces of organs, glands and body cavities. They often metastisize through lymphatic or vascular pathways. Examples include ductal carcinoma which is a type of breast cancer that oriogins from the epithelial lining of breast ducts. Adenocarcinoma which is a type of lung cancer rises from mucus secreting epithelial cells in the lungs.
Mesenchymal tumors arise from mesenchymakl cells from the connective tissue such as bone, cartilage and blood vessels etc. Exampes include:
Osteosarcoma which is a bone cancer originating from osteblasts and liposarcoma which is a malgnancy of adipose tissue.
Hematopoietic tumors: Arise from blood forming or lymphoid tissues such as bone marrow and lymph nodes. They are often systemic. Examples include:
Acute myeloid leukemia a blood cancer originating from myeloid precursors in the bone marrow. Lymphoma which is cancer of lymphoid tissue including hodgkins lymphoma.
Neural tumors originate from the nervous system and can be agressive in their growth and affect functionality of CNS or PNS. Glioblastoma is an agressive brain tumor originating from astrocytic cells in the CNS, and medullablastoma originated from the cerebellum often found in children.
