1. Understanding the cell cycle - chemotherapy and beyond
Julie Mycroft
Principal Pharmacist
Paediatric Oncology
Royal Marsden NHS Trust

2. Cancer treatment modalities
Can be used alone or in combination
Outcome measured in terms of survival rates and response rates

3. Aims of treatment Chemotherapy Hormone Therapy To reduce tumour burden
Improvement in symptoms
Eradication of metastases → cure
Hormone Therapy
Manipulate hormone environment leading to regression of tumours sensitive to hormones
Adjuvant setting
Palliation in advanced disease
Neoadjuvant

4. History of chemotherapy development
1946 Nitrogen mustard given to treat lymphomas
1947 Antifolates introduced
1949 Methotrexate introduced
1950s 5-Fluoro-uracil synthesised
mercaptopurine described
Actinomycin D introduced
1960s Combination chemo cured childhood ALL and HD
Recent Years Many new agents Focus changes to optimising timing and usage and modulating toxicity

5. The cell cycle
aLL

6. Phases of the cell cycle
G0 resting phase
G1 early growth phase
S DNA synthesis
G2 later growth phase
M Mitosis

7. Cell division – mitosis (1)
Prophase
Chromatin condenses into chromosomes. Each chromosome duplicates and consists of 2 sister chromatids. Nucleus breaks down
Metaphase
Chromosomes align and are held by microtubules attached the mitotic spindle and to the centromere

8. Cell division – mitosis (2)
Anaphase
The centromeres divide. Sister chromatids separate and move toward the corresponding poles
Telophase
Daughter chromosomes arrive at the poles and the microtubules disappear. The condensed chromatin expands and the nuclear envelope reappears
The cytoplasm divides, the cell membrane pinches inwards and two daughter cells are produced

9. The Cell Cycle and Tissue Growth
The rate of cell division in human tumours varies considerably from one disease to another
Majority of common cancers increase in size slowly compared to sensitive normal tissues such as BM and GI epithelium
The relationship between cell cycle and cell death affects tumour growth
There is wide variation in the growth rate for various cancers.
Solid tumours usually have a longer doubling time than that of haematological malignancies.
The doubling time for most solid tumours averages about 2-3 months although the range extends from 1 month to several years.
Breast cancer cells, for example, have a doubling time of 100 days, or about 3 months. In contrast, Burkitt’s lymphoma, one of the most rapidly growing haematological malignancies, may demonstrate a doubling time of less then a day.
Actual tumour growth within the body depends on the rate of cell death as well as the rate of cell division. Intially tumour growth is very slow as it has to overcome immunological defence mechanisms in the host, once this is done there is a rapid period of growth where there is a large portion of tumour cells actively dividing and a low rate of cell death. The percentage of actively dividing cells is known as the growth fraction.
The growth fraction decreases as the tumour mass increases in size. A larger tumour mass has a greater percentage of nondividing and dying cells and therefore grows more slowly due to restrictions of space, nutrient availabilty and blood supply to the tumour mass.
This has important implications for the treatment of cancers with chemotherapy as it is most successful in killing tumour cells when the total number of tumour cells is low and the growth fraction is high.
These two conditions occur in the early part of the growth curve and are important reasons for screening programmes for early detection of cancers.
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10. Chemotherapy Effects
Cytotoxic drugs produce their effects by damaging the reproductive potential of cells
The more rapidly growing tumours are more likely to respond to drug treatment
this accounts for leukaemias, lymphomas and testicular cancers being more responsive than colonic / pancreatic cancers
Most cytotoxic chemotherapy drugs damage cancer cells by either interfering with the synthesis of the precursors of DNA or chemically interacting with the DNA itself to interfere with the process of cellular division.
As discussed previously the more rapidly growing tumours are more likely to respond to drug treatment which accounts for leukaemias, lymphomas and testicular cancers being more responsive than colonic/pancreatic cancers.
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11. Growth Fraction
At a given time, the number of cells in a population that are actively passing through the cell cycle divided by the total number of cells in the population = growth fraction
The greater the growth faction, the more likely the treatment will produce cell death
Most cytotoxic chemotherapy drugs damage cancer cells by either interfering with the synthesis of the precursors of DNA or chemically interacting with the DNA itself to interfere with the process of cellular division.
As discussed previously the more rapidly growing tumours are more likely to respond to drug treatment which accounts for leukaemias, lymphomas and testicular cancers being more responsive than colonic/pancreatic cancers.
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12. Kinetics of cell killing
Fractional Cell kill hypothesis
A given dose of cytotoxic drug kills a given proportion of cells, not a given number
Smaller tumours require fewer cycles of chemotherapy than larger ones
Pulsed intermittent therapy
Maximises tumour cell killing whilst allowing normal tissues damaged by the drug to recover

13. Cytotoxic Drug Classification
Cell Cycle Phase-Specific Agents
active in a particular phase of cell cycle
Depend on the production of some type of unique biochemical blockade of a particular reaction occurring in a single phase of the cell cycle
Cell Cycle Phase-Non-specific Agents
Cytotoxic effect exerted irrespective of cell cycle state
equally effective in large tumours in which cell growth is low
dose dependent
single dose has same effect as repeated fractions totalling the same amount
Cytotoxic chemotherapy agents have traditionally been classified as phase- or non-phase specific, depending on the effect on the cell cycle.
In vitro models demonstrate that phase dependant drugs kill cells exponentially at lower doses but reach a plateau when given at a higher dose because they are only able to kill cells in a specific part of the cycle. They are most effective against tumour which have a large proportion of actively dividing cells.
Greater cell kill is achieved if they are given in multiple repeated fractions.
Non phase dependant drugs are equally toxic for both cycling cells and those in G0 therefore are effective in large tumours where cell growth is low.
They kill cells exponentially with increasing dose therefore they are dose dependant. A single dose has the same effect as repeated fractions totalling the same amount.
Draw on any experience to give examples of phase and non-phase specific agents – refer to separate photocopied sheet Figure 3-4 cell specificity of anticancer drugs
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14. Phase Specificity of Cytotoxic Drugs
Carry on from discussion with this slide which gives which phase of the cycle chemotherapy agents work at
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(which moves onto specific modes of actions for each group of cytotoxics)

15. Mechanisms of Action (1)
Alkylating agents and nitrosureas
Highly reactive molecules
Interfere with replication by covalently linking an alkyl group (R-CH2+) to nucleic acids and proteins of the base pairs of the cellular DNA
causes the strands of DNA to cross-link either within a strand or between strands.
Mechanism of toxicity - impairment of DNA replication
Examples
e.g cyclophosphamide, chlorambucil, melphalan, nitrosoureas eg carmustine
alkylator-like agents - cisplatin, carboplatin, procarbazine, dacarbazine
The alkylating agents were the first compounds identified to have activity against neoplastic diseases and have been in use now for over 50 years.
Alkylating agents undergo transformation to produce highly reactive, positively charged ions therefore they are electron deficient. These ions can then form covalent bonds with electron rich sites on biological molecules such as nucleic acids, proteins, and amino acids.
The process of substitution of an alkyl group for a hydrogen atom is referred to as alkylation and this seems to be the predominant cause of cell death.
There are 3 possible outcomes of this alkylation process
The template, which is the DNA strand being replicated, may be misread or mispaired during DNA synthesis
Cross-linking may prevent the DNA strands from unwinding which would hinder the reolication process
The alkylating agents may produce either single- or double strand breaks in the DNA
Any of these actions inhibits the synthesis of DNA, RNA or protein
Alkylating agents have a steep dose response curve which means that the greater the dose of drug the greater the fraction of cell kill – this makes them useful in dose intensification strategies.
Within the alkylating group are the classic alkylators such as cyclophosphamide, chlorambucil, melphalan, the nitrosureas such as carmustine and lomustine and alkylator like agents such as cisplatin, carboplatin, procarbazine and dacarbazine
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17. Mechanisms of Action (2)
Antitumour Antibiotics
Disrupt normal replication by binding to DNA intercalating between the base pairs blocking the transcription of DNA
Breaks in DNA may also occur
Examples
Anthracyclines ie Doxorubicin Epirubin, Mitoxantrone, Actinomycin D Bleomycin,

18. Mechanisms of Action (3)
Antimetabolites
Interfere with normal synthesis of nucleic acids
Cell cycle phase specific (S phase)
folate antagonist - MTX
pyrimidine antagonists - cytarabine, 5-fluorouracil, capecitabine, gemcitabine
purine antagonists - cladribine, mercaptopurine, thioguanine, fludarabine
adenosine deaminase inhibitor - pentostatin
Antimetabolites are separated into
folate antagonists – methotrexate (reduced folate is required for synthesis of purine and pyrimidine, folate is reduced by the enzyme dihydrofolate reductase (DHFR) – MTX binds to and inhibits DHFR
pyrimidine antagonists – cytarabine, fludarabine, 5FU and gemcitabin
purine antagonists – cladrabine, mercaptopurine, thioguanine and fludarabine
adenosine analogs or adenosine deaminase inhibitors – pentostatin and cladrabine
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19. Mechanisms of Action (4)
Disrupt the M phase of the cell cycle
Vinca alkaloids
Inhibit the assembly of microtubules by binding to tubulin resulting in the dissolution of the mitotic spindle required for chromosome division
e.g vincristine, vinblastine, vinorelbine
Taxanes
Bind to stabilised microtubules once they have formed, resulting in arrest of normal mitotic cell division and subsequently cell death
e.g paclitaxel, docetaxel

20. Mechanisms of Action (5)
Camptothecans
Inhibit type I DNA topoisomerase.
Act predominantly in the S phase
e.g topotecan, irinotecan
Epipodophyllotoxins
Inhibit type II DNA topoisomerase and prevent cells from entering mitosis
Produce protein-associated DNA double strand breaks
e.g etoposide

21. Mechanisms of Action (6) – Misc. Asparaginase
Normal Cell
Tumor Cell
Aspartic Acid
+ L-Glutamine
L-Asparagine
Synthetase
L-A´ase
L-Asparagine
(cell produced)
L-Asparagine
Deficiency
Aspartic Acid +
Ammonia
Cell Proliferation
Cell Death

22. Combination Chemotherapy (1)
Early studies used single agents, but remissions were short and relapse was associated with drug resistance
Combination chemotherapy is used to try and improve rate and duration of response by combining drugs with different mechanisms of action. This also helps prevent resistance mechanisms
Despite knowledge of cell kinetics, most regimens have been decided on empirically
During the 1950’s drugs were mainly used as single agents and yeilded only a few complete responses with short durations – it is limited by the development of acquired resisitance.
To increase the likelihood of affecting more clones and killing more tumour cells it has become customary to combine several agents that that have activity against a particular tumour type when developing a regimen.
Despite a knowledge of cell kinetics most drug schedules have been decided by a knowledge of efficacy, toxicity and practicality
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23. Combination Chemotherapy (2)
The combination of drugs is chosen based on some common principles
Use drugs that are known to be effective as single agents
Use drugs with non-overlapping toxicity
pulsed intermittent therapy should be used to allow the GIT and the bone marrow to recover
Use the optimal dose and schedule for each individual agent
If possible use drugs with synergistic killing effects
Use drugs which work at different phases in the cell cycle
Follow schedules that are supported by experience or observation, not just theory
The development of a combination chemotherapy schedule should follow a number of general principles as seen here:
Refer to principles on slide
Make comments regarding:
Synergistic killing – not normally known in practice, one example is the use of 5FU and folinic acid
Informed empiricism – alkylating agents are given before antimetabolite. This is because in a resting cell DNA damage caused by an alkylating agent can be repaired by the time the cell enters the cycle – by giving the antimetabolite after these repaired cells can be damaged in the S phase.
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(which moves onto the new and last topic of drug interactions)

24. Short-term toxicity Haematopoietic System Gastro-intestinal tract
Bone marrow suppression occurs when the pool of stem cells has been damaged by chemotherapy.
The store of mature blood cells in the bone marrow lasts for around 8 to 10 days following treatment, after which leucopenia and thrombocytopenia can develop
Gastro-intestinal tract
Nausea and vomiting are common in patients treated with intravenous alkylating agents, doxorubicin and cisplatin.
Hair loss
Scalp cooling may be used

25. Long term toxicity Impaired gonadal function Pulmonary fibrosis
Procarbazine and alkylating agents
men – decreased spermatogenesis
women – ovarian failure
Pulmonary fibrosis
Busulfan, Bleomycin
Organ damage
Liver damage – antimetabolites
Cardiac damage – anthracyclines
Second Cancers
Alkylating agents, etoposide, anthracyclines

26. Drug Resistance
Cells in a solid tumour are not uniformly sensitive to a cytotoxic drug
As the tumour grows, greater heterogeneity develops and cell mutation occurs
Host defence mechanisms and the use of cytotoxic drugs exert a selection pressure encouraging the survival of the resistant cells , which grow and multiply
Despite the notable advances in cancer management over recent decades drug resistance remains the most important reason for treatment failure.
Now going to cover some of the aspects relating to drug resistance.
The cells in a solid tumour are not uniformly sensitive to a cytotoxic drug before treatment starts.
It has been suggested that genetic instability increases as cancers grow and that greater heterogeneity develops accordingly. This concept provides a partial explanation for greater drug resistance in large tumours (as judged by diminished extent and duration of response)
As the tumour grows in size the frequency of development of resistant cells increases so that large tumours have a greater number of intrinsically resistant cells. This resistance is produced by one or more mechanisms which I will go into in a bit more detail in a moment.
Host defence mechanisms and the use of cytotoxic drugs exert a selection pressure encouraging the survival of resistant cells which grow and multiply.
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27. Cellular Mechanisms of Resistance
Mechanism Drug
Efficient repair to damaged DNA alkylating agents
Decreased uptake by cell MTX, doxorubicin
Increased drug efflux epipodophyllotoxin
(p-glycoprotein) vincs, anthracyclines
Decreased intracellular activation 6MP,5-FU
Increased intracellular breakdown cytarabine
Bypass biochemical pathways MTX, 6MP, asparaginase
Gene amplification or over- MTX, nitrosoureas
production of blocked enzyme
So these mechanisms in a bit more detail.
The degree of DNA damage caused by a drug depends on the balance of the rate of damage and the rate of repair. There are certain nuclear proteins that recognise DNA damage and as a result DNA can be repaired by a series of enzymatic steps eg with alkylating agents the damaged base is recognised, excised and the resultant gap repaired using the other DNA strand as a template.
If a drug enters the cell by a specific active transporter protein, cells can become resistant if the activity of the transport system is decreased eg MTX enters via the reduced folate transporter protein and uses a second folate transporter protein that doesn’t bind MTX – this results in 250 fold higher concentration of MTX being needed.
Cells can become resistant to drugs that enter cells by passive diffusion, namely hydrophobic compunds. This can result in a cell having multiple drug resistance (MDR). Several proteins that function to transport drugs and other molecules out of cells can give rise to MDR. The main protein described is p-glycoprotein. Patients whose tumours express P-gp at presentation are three times more likely to fail to respond to chemotherapy. Patients with P-gp +ve tumours after therapy do even worse having a fourfold increase in the rate of treatment failure.
Cont………..

28. Other Mechanisms of Drug Resistance
Diminished vascularity
Only a small proportion of cells may be in cycle, allowing time for repair from cytotoxic damage before cell division
In addition to all these types of cellular resistance at least 2 other mechanisms are important.
The first is diminished vascularity of parts of the tumour as it becomes larger, resulting in hypoxia and decreased drug penetration.
The second is that only a small proportion of cells may be in cycle, allowing for repair from cytotoxic damage before cell damage.
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(Which moves onto the new topic of chemo combinations)

29. Hormone Therapy
Beatson demonstrated that some inoperable breast cancers regressed after removal of the ovaries (oophorectomy)
Many years later Huggins showed that metastatic prostatic cancer regressed after removal of the testes (orchidectomy).
In breast cancer, hormone receptor status is clinically important in management

30. Principals of hormone therapy (1)
Receptor proteins for steroid hormones are found in both the cytoplasm and the nucleus
The interaction between these hormones and their receptor proteins promotes cell growth and division.
The steroid hormone crosses the cell membrane and forms a complex with a receptor in the cytoplasm.
This activated complex passes into the nucleus where it binds to a protein, leading to the production of messenger RNA (mRNA) and then protein.
Finally DNA is synthesised and the cell divides

31. Principals of hormone therapy (2)
This interaction provides the rationale for a number of ways in which hormone manipulation can modify tumour growth. It may be possible to:
lower the plasma concentration of hormone by removing the source of production, e.g. the testes or ovaries
prevent the hormone from binding to receptor via competitive inhibition or by reducing synthesis of the receptors
block binding of the hormone/receptor complex to DNA in the nucleus
The precise mode of action of agents used in hormone therapy is often unclear.

32. Approaches to hormone therapy (1)
Lowering plasma hormone concentration
Radiotherapy Radiotherapy to the ovaries induces the menopause – the ovaries stop producing eggs and the female sex hormones.
Surgery In breast cancer can involve:
the ovaries (oophorectomy)
the adrenals (adrenalectomy)
the breast tissue (mastectomy)
In prostate cancer:
surgical removal of the testes (orchidectomy)
Medical treatment Aromatase inhibitors
Analogues of luteinizing hormone–releasing hormone (e.g. goserelin and leuprorelin)

33. Approaches to hormone therapy (2)
Blocking the action of circulating hormones
Anti-oestrogens and anti-androgens work by blocking the binding of hormones to their receptors.
Anti-oestrogens (e.g. tamoxifen)
Anti-androgens (e.g. flutamide, megestrol acetate)
Additive hormone therapies
The action of circulating hormones can also be blocked by additive hormone therapies, which in breast cancer include oestrogens, androgens, glucocorticoids and progestogens.

34. Summary
A knowledge of the cell cycle is important to understanding the mechanism of action of cancer chemotherapy
Combination chemotherapy is used to try and improve rate and duration of response by combining drugs with different mechanisms of action.
Manipulating the interaction between hormones and cell growth provides a means for treating hormone sensitive cancers

35. Acknowledgements
The Institute of Cancer Research - interactive education unit module 4 “An approach to therapies”
‘Cancer and it’s Management’ - 3rd Edition Souhami R and Tobias J (Blackwell Science)