Chemotherapy Shamsun N. Khan, Ph.D, Postdoc Harvard, USA 1

Chemotherapeutic Agents Most effective cytotoxic agents damage DNA Toxicity is greater in the S or DNA synthetic, phase of the cell cycle. Vinca alkaloids and taxanes, block formation of a functional mitotic spindle in M phase Human neoplasms that are most susceptible to chemotherapy are those with a high percentage of cells under going division. 2

The nucleus is a membrane bound organelle that contains the genetic information in the form of chromatin, highly folded ribbon-like complexes of deoxyribonucleic acid (DNA) and a class of proteins called histones. 3

Histone with DNA 4

Cell Divisions through Microscope When a cell divides Chromatin fibers are very highly folded visible in the light microscope as chromosomes. During interphase (between divisions), chromatin is more extended, a form used for expression genetic information. 5

Cell Cycle Regulation Regulation of the cell cycle How cell division (and thus tissue growth) is controlled is very complex. Cancer is a disease where regulation of the cell cycle goes awry and normal cell growth and behavior is lost. 6

Cdk Cdk (cyclin dependent kinase, adds phosphate to a protein), along with cyclins, are major control switches for the cell cycle, causing the cell to move from G1 to S or G2 to M. 7

G1 is the gap period between mitosis and the beginning of DNA synthesis 8

MPF (Maturation Promoting Factor) includes the CdK and cyclins that triggers progression through the cell cycle. 9

p53 Gene p53 is a protein that functions to block the cell cycle if the DNA is damaged. If the damage is severe this protein can cause apoptosis (cell death). p53 levels are increased in damaged cells. This allows time to repair DNA by blocking the cell cycle. Gives the cell rest. 10

Danger!!!! p53 mutation!!! A p53 mutation is the most frequent mutation leading to cancer. An extreme case of this is Li Fraumeni syndrome, where a genetic a defect in p53 leads to a high frequency of cancer in affected individuals. 11

P27 Protein p27 is a protein that binds to cyclin and cdk blocking entry into S phase. Nature Medicine 3, 152 (1997)) suggests that breast cancer prognosis is determined by p27 levels. Reduced levels of p27 predict a poor outcome for breast cancer patients. 12

What is (and is not) mitosis? Mitosis is nuclear division plus cytokinesis, and produces two identical daughter cells during prophase, prometaphase, metaphase, anaphase, and telophase. Interphase is often included in discussions of mitosis, but interphase is technically not part of mitosis, but rather encompasses stages G1, S, and G2 of the cell cycle. 13

The cell is engaged in metabolic activity and performing its prepare for mitosis (the next four phases that lead up to and include nuclear division). Prophase 14

The nuclear membrane dissolves, marking the beginning of prometaphase. Proteins attach to the centromeres creating the kinetochores. Prometaphase 15

Metaphase Spindle fibers align the chromosomes along the middle of the cell nucleus. This line is referred to as the metaphase plate. This organization helps to ensure that in the next phase, when the chromosomes are separated, each new nucleus will receive one copy of each chromosome. 16

Anaphase The paired chromosomes separate at the kinetochores and move to opposite sides of the cell. Motion results from a combination of kinetochore movement along the spindle microtubules and through the physical interaction of polar microtubules. 17

Chromatids arrive at opposite poles of cell, and new membranes form around the daughter nuclei. The chromosomes disperse and are no longer visible under the light microscope. The spindle fibers disperse, and cytokinesis or the partitioning of the cell may also begin during this stage. Telophase 18

Cytokinesis In animal cells, cytokinesis results when a fiber ring composed of a protein called actin around the center of the cell contracts pinching the cell into two daughter cells, each with one nucleus. In plant cells, the rigid wall requires that a cell plate be synthesized between the two daughter cells. 19

DNA or deoxyribonucleic acid is a large molecule structured from chains of repeating units of the sugar deoxyribose and phosphate linked to four different bases abbreviated A, T, G, and C. We will later show how the simple structure of DNA contains the information for specifying the proteins that allow life. The process of mitosis is designed to insure that exact copies of the DNA in chromosomes are passed on to daughter cells. 20

Alkylating Agents (1) the nitrogen mustards (2) the ethyleneimines (3) the alkyl sulfonates (4) the nitrosoureas and (5) the triazenes 21

Pharmacological Actions Alkylating agents disturb DNA synthesis and cell division. (Dividing Cells) Interfere with DNA integrity and function to induce cell death in rapidly proliferating tissues provides the basis for their therapeutic and toxic properties. 22

Cytotoxic Effect on Low Mitotic indices Certain alkylating agents may have damaging effects on tissues: Examples are as follows: Liver, kidney, and low mitotic indices: mature lymphocytes—effects in these tissues usually are delayed. 23

Mitotic index can be calculated using the following operation : cells observed with visible chromosomes ÷ total number of cells visible 24

Cell counting is one of the most important tools for cell biologists. Cell counts are routinely used to determine cell culture health and the effect of drugs or toxins. However, cell counting is invasive and time consuming. Cells need to be detached from their growing surface, removed from the cell culture vessel and possibly stained prior to counting. 25

The HoloMonitor non-destructively counts the number of adherent cells directly in a cell culture vessel by counting cells in a sufficient number of randomly selected images of the cell culture. 26

Acute/Delayed Effects Acute effects are primarily against rapidly proliferating tissues. Lethality of DNA alkylation depends on recognition of the adduct, creation of DNA strand breaks by repair enzymes, and an intact apoptotic response. 27

DNA Damage in Non-dividing Cells DNA damage activates a checkpoint that blocks cell-cycle progression at the G1/S interface. Allowing cells to either repair DNA alkylation or undergo apoptosis. Malignant cells with mutant or absent p53 fail to suspend cell-cycle progression, do not undergo apoptosis, and are resistant to these drugs. 28

Distinction between Monofunctional and Bifunctional Agent DNA is the ultimate target of all alkylating agents, there is a crucial distinction between the bifunctional agents, in which cytotoxic effects predominate: Monofunctional methylating agents (procarbazine, temozolomide), which have greater capacity for mutagenesis and carcinogenesis. 29

Cross-linking of DNA strands is a much greater threat to cellular survival than Single-base alkylation and the resulting depurination and chain scission. 30

Frequent Methylation By passed by DNA polymerases, Leading to mispairing reactions that permanently modify DNA sequence. These new sequences are transmitted to subsequent generations, and may result in mutagenesis or carcinogenesis. Methylating Agents: procarbazine, are highly carcinogenic. 31

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Mechanisms of Resistance to Alkylating Agents 1.Decreased permeation of actively transported drugs (mechlorethamine and melphalan). 2. Increased intracellular concentrations of nucleophilic substances, principally thiols such as glutathione, which can conjugate with and detoxify electrophilic intermediates; 33

3. Increased activity of DNA repair pathways-may differ for the various alkylating agents. 4. Increased activity of the complex nucleotide excision repair (NER) pathway: seems to correlate with resistance to most chloroethyl and platinum adducts. 34

5. Alkyl guanine transferase (AGT) activity determines response to BCNU and to methylating drugs such as the triazenes, procarbazine, and busulfan 6. Increased rates of metabolism of the activated forms of cyclophosphamide and ifosfamide to their inactive keto and carboxy metabolites by aldehyde dehydrogenase. 35

TOXICITY 36

Bone Marrow Toxicity Most alkylating cause dose-limiting toxicity to bone marrow and intestinal mucosa. alkylating agents, including nitrogen mustard, melphalan, chlorambucil, cyclophosphamide, and ifosfamide, cause acute myelosuppression, 37

Peripheral blood granulocyte count at 6– 10 days and recovery in 14–21 days. Cyclophosphamide has lesser effects on peripheral blood platelet counts than do the other agents. Busulfan suppresses all blood elements, particularly stem cells, and may produce a prolonged and cumulative myelosuppression lasting months or even years. 38

For this reason, it is used in preparation for allogeneic bone marrow transplantation. Carmustine and other chloroethylnitrosoureas cause delayed and prolonged suppression of both platelets and granulocytes After 4–6 weeks drug administration and reversing slowly thereafter. 39

Both cellular and humoral immunity are suppressed by alkylating agents, These have been used to treat various autoimmune diseases. Immunosuppression is reversible at doses used in most anticancer protocols. 40

Mucosal Toxicity Alkylating agents are highly toxic to dividing mucosal cells, leading to oral mucosal ulceration and intestinal denudation. The mucosal effects are particularly significant in high-dose chemotherapy 41