Cancers and Identity Based Testing

Cancers

  • Cancer develops when cells grow uncontrollably due to tumor-promoting agents.
  • Major changes for a cell to become cancerous:
    • Immortalization: Unregulated cell division without regard to cellular regulators.
    • Transformation: Upregulation (gain of function) or downregulation (loss of function) of normal proteins.
      • Increased angiogenesis: Creation of new blood vessels to supply nutrients to tumor cells.
    • Metastasis: Ability of cancer cells to migrate from the initial tumor site to other organs.
      • Example: Prostate cancer cells metastasizing to bones and lungs.

Genetic Changes in Tumorigenesis

  • Activation of proto-oncogenes to oncogenes.
  • Inactivation of tumor suppressor genes.
Proto-oncogenes and Oncogenes
  • Proto-oncogenes: Normal genes involved in cell growth and proliferation.
  • Oncogenes: Activated proto-oncogenes that promote tumorigenesis.
    • Promote cell division and inhibit apoptosis.
  • Inappropriate activation of proto-oncogenes can be due to:
    • Qualitative changes:
      • Deletion or point mutation in coding sequences.
    • Quantitative changes:
      • Retroviral insertion: Insertion of a retroviral promoter activates proto-oncogenes.
      • Chromosomal translocation: Gene fusion near proto-oncogenes leads to activation.
      • Gene amplification: Increased production/expression of genes.
  • Activation of proto-oncogenes to oncogenes results in a gain of function, leading to increased cell division and proliferation, or regulated apoptosis.
Examples of Proto-oncogenes and Oncogenes
  • CRSC Tyrosine Kinase:
    • The CRSC gene codes for tyrosine kinase, which transfers phosphate from ATP to tyrosine on target proteins (acts as a phosphorylase).
    • Increased CRSC production leads to unregulated cell signalling, resulting in increased cell division and proliferation.
    • Inactive kinase has a closed conformation; activated kinase binds substrates, leading to phosphorylation and increased signalling.
  • CMYC:
    • Encodes a helix-loop-helix transcription factor.
    • Forms a heterodimer with MAX protein to regulate gene expression.
    • CMYC regulates apoptosis and genes involved in cell cycle regulation.
    • Increased CMYC production leads to decreased apoptosis and unregulated cell cycle.
Inactivation of Tumor Suppressor Genes
  • Tumor suppressor genes control transcription/translation of genes required for cell division.
  • Inactivation leads to a loss of function, promoting increased cell division.
  • Normally, these genes slow down or stop cell division at checkpoints. Some arrest the cell cycle at specific phases.
  • Cancer arises when two independent mutations inactivate both tumor suppressor genes at a locus.
Examples of Tumor Suppressor Genes
  • Retinoblastoma Protein (PRB):
    • A cell cycle "master switch."
    • Normally, PRB binds to the E2F transcription factor.
    • Binding inactivates E2F, leading to loss of translation of certain genes.
    • During the S phase, normally E2F is released from PRB and binds to certain genes
    • PRB is regulated by cyclin-dependent kinases (cyclin D and cyclin E).
    • Phosphorylation of PRB releases E2F allowing expression of certain genes.
    • Inactivated PRB results in unregulated binding of E2F to genes, with unregulated protein production occurring in S phase, G2, M, and G1 phases.
    • Hypophosphorylated PRB binds to E2F, preventing it from binding to target genes.
  • Knudson's Two-Hit Hypothesis:
    • Proposed by Dr. Alfred G. Knudson based on observations of inherited vs. non-inherited tumors.
    • Two mutations/hits are required to activate the cancer-causing gene.
    • Inheriting one mutation is not sufficient; both alleles must be mutated.
  • P53:
    • Referred to as the "guardian of the genome."
    • Regulates DNA damage control.
    • 80%80\% of human cancers show deletion of both alleles on chromosome 17.
    • Functions:
      • Stops cell cycle progression to S phase (G1 arrest).
        • In DNA damage cases, P53 is activated and binds to DNA encoding P21 cyclin-dependent kinase.
        • P21 inhibits cyclin-dependent kinases 4 and 2, leading to G1 arrest.
      • Inhibits cell cycle progression to M phase (G2 arrest), leading to apoptosis.
    • Decreased P53 production leads to unrestrained cell growth.
    • Two possible causes:
      • Inactivation of P53.
      • Production of mutant P53.
    • Mutant P53 usually leads to unrestrained cell growth.
    • P53 stimulates G1 arrest (preventing S phase) and facilitates G2 arrest (leading to apoptosis).

Chromosomal Rearrangement

  • Many cancers are associated with chromosome alterations, particularly translocations.
  • Translocation can bring a proto-oncogene under the control of the wrong promoter.
  • Examples:
    • Burkitt's Lymphoma:
      • Translocation from 8 to 14, denoted as t(8;14).
      • Activates CMYC.
    • Chronic Myelogenous Leukemia (CML):
      • Translocation from 9 to 22, denoted as t(9;22).
      • Results in the Philadelphia chromosome.
      • Fuses BCR and ABL proto-oncogene, generating BCR-ABL fusion protein with unregulated tyrosine kinase activity.
    • Acute Promyelocytic Leukemia:
      • Translocation from 15 to 17.
      • Fuses PML and RAR, inhibiting P53.

Viruses and Cancer

  • Tumor cells can arise through the action of tumor viruses.

  • Viruses classified cells into permissive and non-permissive cells

  • Classified into two types, DNA tumor viruses and RNA tumor viruses

  • DNA Tumor Viruses:

    • Hepatitis B
    • Human Papilloma Virus
    • Epstein-Barr Virus

  • RNA Tumor Viruses:

    • Human Immunodeficiency Virus 1
    • Human T-cell Leukemia Virus 1
Hepatitis B Virus (HBV)
  • Causes liver cirrhosis, which can lead to hepatocellular carcinoma.
  • 70%70\% of HBV-associated hepatocellular carcinomas produce HBVX protein.
  • HBVX protein is important for carcinogenesis, facilitating dysregulation in cellular processes.
    • Promotes transcription factor activation through the RASRAF MAPK pathway, leading to increased mitogen production and cell division.
    • Facilitates transcriptional activation of proto-oncogenes (CMYC, CSRC) and represses tumor suppressor genes (P21, P27), causing deregulation of gene expression.
    • HBX binds to DDB1 (important for ubiquitination), leading to degradation of many proteins.
    • Increases production of cccDNA (DNA produced by liver cells hijacked by HBV), which leads to production of pgRNA (important for HBV replication).
Human Papilloma Virus (HPV)
  • Small, non-enveloped viruses with dsDNA genomes.
  • Wart-causing viruses that infect basal epithelial cells; continued expression of viral E6 and E7 genes.
  • E7:
    • Inactivates tumor suppressor gene PRB, leading to unregulated binding of E2F and increased cell division.
  • E6:
    • Binds to ubiquitin protein ligase and P53, leading to P53 degradation.
    • Binds to CMYC, leading to increased production of HTERT (human telomerase reverse transcriptase), which increases telomerase activity, leading to cellular immortality.
  • Telomeres maintained by telomerase; telomere shortening during cellular aging.
  • Telomerase activity usually observed only in stem cells; increased telomerase activity leads to cellular immortality.

Retroviruses

  • Can transform cells through two mechanisms:
    • Introduction of oncogenes (e.g., v-SRC, v-MYC).
    • Retroviral promoter/enhancer insertion, activating proto-oncogenes to oncogenes.

Chemical Carcinogenesis

  • Some carcinogens act by genotoxic or non-genotoxic mechanisms.

Identity Based Testing

  • Techniques to determine the identity of specific humans/suspects for forensics, paternity, sibling tests, bone marrow engraftment testing.
  • DNA typing is reliable and conclusive for identification.

DNA Polymorphisms

  • Variations in DNA sequences shared by a percentage of the population.
  • Types:
    • Single Nucleotide Polymorphisms (SNPs): Unique per individual; contribute to human uniqueness.
    • Variable Number Tandem Repeats (VNTRs) and Short Tandem Repeats (STRs): Copy number variants.
      • Siblings share some STRs.
Restriction Fragment Length Polymorphism (RFLP)
  • Original DNA targets used in gene mapping, human identification, and parentage testing.
  • Observed as differences in the fragment sizes and numbers generated by restriction enzyme digestion of DNA.
  • Targets VNTRs; restriction enzymes target sequences found in VNTRs.
  • Southern blot is the method for detection and analysis of RFLP.
Parentage Testing with RFLP
  • Alleged fathers are identified based on the ability to provide the remaining alleles.
  • Shared allele/obligate paternal allele: Allele found in the child that is also found in the father.
Fingerprinting with RFLP for Forensic Analysis
  • Compare migration patterns of evidence samples and suspect DNA.
  • Similar migration patterns suggest a match.
  • Qualitative approach; largely replaced by STR analysis.
Short Tandem Repeats (STR) Analysis
  • Microsatellites are tandem repeats ranging from 2-6 base pairs distributed throughout the body.
  • Less laborious; uses software to check for STR loci.
  • Uses PAGE and capillary gel electrophoresis.
  • Small sample volume required.
  • In the US, 13 STR loci are selected for human identification, serving as allele ladders.
  • STRs are part of copy number variations.
  • The chosen STRs produce lower stutter rates and mutation rates.
  • STR analysis can be done via multiplex PCR, where many losses are targeted at once. It produces allelic ladders.
  • To do it, a DNA profile is constructed for a sample and compared to query sample
STR Analysis for Paternity Testing
  • Uses 13 tetranucleotide STR loci.
  • Software calculates the paternity index for each locus in which the alleged father and child share an allele.
  • Paternity index: How many times more likely the child's allele is inherited from the alleged father than another man.
  • If there are no shared alleles, the paternity index is zero.
  • Combined Paternity Index (CPI): Product of all paternity indices.
  • Probability of Paternity: Probability that the alleged father is the source of the paternal genes found in the child.
    • Calculated from CPI and prior odds (assumed to be 50%50\%.
    • Formula: CPI×0.50CPI×0.50+(1−0.50)\frac{CPI \times 0.50}{CPI \times 0.50 + (1 - 0.50)}
    • Higher probability indicates a higher chance the alleged father is the true father.
STR Analysis for Bone Marrow Transplantation
  • Used to check for the success of bone marrow engraftment.
  • Bone marrow transplantation treats malignant and non-malignant blood disorders.
  • Allogeneic transplant strategy: Donor cells come from a separate, highly compatible relative.
  • Submyeloablative procedure removes recipient bone marrow, then donor bone marrow is transplanted.
  • Successful engraftment leads to a genetic chimera (patient contains two types of cells from different stem cell lineages).
  • Pre-transplant testing:
    • Screens for informative loci (loci that help differentiate between donor and recipient cells).
  • Post-transplant testing:
    • Determines the quantity of donor cells relative to recipient cells.
    • Full chimerism: 95-99% of recipient bone marrow contains donor cells.
    • Mixed chimerism: >10% but <95% of recipient bone marrow contains donor cells.
    • Graft failure: <5-10% donor cells (recipient immune system rejects donor cells).
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