Strategy (ies) in Cell Cycle ResearchAleš Hampl

DNA Replication

Nuclear division

Cytokinesis

Sequentially occur in every cell division

Cell cycle that contains G1, S, G2, and M phases

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Two aspects of the word „Strategy“:

1) Approaches used to uncover the molecular mechanisms involved in regulation of the progression of cell cycle (in general terms)

2) Important biological phenomena that are linked to the progression of cell cycle (morphogenesis, cancer,…)

Aspect no. 1 - Approaches

Little bit of not that long history(~3 decades)

80ties of the last century = beginnings of cell cycle research

1971 - first description of the cytoplasmic activity called „MPF“ in meiotically maturing frog oocytes (Masui and Markert, J. Exp. Zool.)

MP

F a

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G2-arrested oocyte oocyte at MI

oocyte at interphaseMII-arrested egg

zygote at interhase

progesteron sperm penetration

„MPF assay“

GVBD

The existence of MPF is not restricted only to maturing oocytes but it is present in a wide variety of cells:

1978 – cleaving embryos (Wassermann and Smith, J. Cell Biol.)

1979 – mammalian cultured cells (Sunkara et al., Proc. Natl. Acad. Sci. USA)

1982 – yeast Saccharomyces cerevisiae (Weintraub et al., CR Acad. Sci. Paris)

Properties of MPF are the same in all cell types assayed:

• MPF is temporally correlated with M phase• MPF promotes the entry into M phase• when MPF activity declines, nuclei reform, chromosomes decondense, and DNA synthesis begins

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All eukaryotes use the same „principle“ to coordinate

progression of their cell cycle = their cell division

1980, 1981 – it was shown the major role of the product of cdc2 gene in controlling the cell cycle in the fission yeast Schizosaccharomyces pombe

(Nurse and Thuriax, Genetics; Nurse and Bisset, Nature)

Temperature sensitive mutants of the cdc2 gene become arrested either in G1 before S-phase or in G2 before mitosis, when S. pombe is incubated at nonpermissive temperature. S. pombe cdc2 gene has its homolog CDC28 in Saccharomyces cerevisiae.

Important lesson learned from yeast:

1985 – 1990 = golden years of cell cycle research

1985, 1986 – CDC28 gene of S. cerevisiae and cdc2 gene of S. pombe were shown to encode a protein kinase

potentially regulated by phosphorylation (Reed et al., Proc. Natl. Acad. Sci. USA; Simanis and Nurse, Cell)

• both cdc2 and CDC28 genes encode for 34/36 kD phosphoprotein with protein kinase activity (62% amino acid sequence identity)• the level of p34/p36 protein remains unchanged in proliferating cells• when cells cease to proliferate and arrest in G1 the p34/p36 protein becomes dephosphorylated and looses its kinase activity

Yeast was the first:

Vertebrates also have it:

1987 – human homolog of cdc2 gene was identified by functional complementation of the S. pombe cdc2 mutation

(Lee and Nurse, Nature)

1987 – identification of p34 protein in human cells that is homologous to p34cdc2 protein of S.pombe and p36CDC28 of S. cerevisiae

(Draetta et al., Cell) Human p34 protein was immunoprecipitated from [35S]methionine labeled HeLa cells using monoclonal antibody raised against p34cdc2 crossreacting with p34CDC28.

Link between p34cdc2 and MPF becomes established:

1988 – MPF isolated (by affinity chromatography using p13suc1) from frog oocytes is composed from two major components, proteins of molecular weights 34 kD and 42 kD, with p34 most likely being homolog of p34cdc2

(Dunphy et al., Cell, July 29)

1988 – MFP purified from frog oocytes contains 32 kD protein that

is recognized by antibody against p34cdc2; 32 kD protein is associated with 42 kD protein thus creating a complex that expresses protein kinase activity

(Gautier et al., Cell, July 29)

1988 – when purified from starfish oocytes, „M phase-specific“ histone H1 kinase (H1K) activity coelutes with 34 kD protein that crossreacts with antibody against p34cdc2; MPF and H1K appear to be the same entity

(Arion et al., Cell)

• p34 is unphosphorylated, unassociated with p62 protein, and inactive as kinase in G1 cells• each of these conditions is reversed in G2/M cells• in cells arrested in metaphase by exposure to nocodazole, the p34/p62 complex accumulates in an active state

1988 – human p34 (HeLa cells) undergoes cell cycle-dependent phosphorylation and subunit rearrangement

(Draetta and Beach, Cell)

Biochemistry of human p34 suggests its role in cell cycle regulation:

1983 – described for the first time as the protein that undergoes repeated destruction in cleaving

sea urchin eggs (Evans et al., Cell)

1989 – cyclin synthesis is necessary for entry into mitosis in frog eggs (Minshull, Blow, and Hunt; Cell)

1989 – destruction of cyclin is required for exit from mitosis in early frog embryos

(Murray, Solomon, and Kirschner; Nature)

Cyclin comes to play:

Example: fluctuation of cyclins A and B in early clam embryos

minutes after fertilization

cyclin A

cyclin B

Westendorf et al., J. Cell Biol., 1989

1990 – cyclins B1 and B2 are the components of MPF from frog oocytes

(Gautier et al., Cell)• immunoprecipitates of cyclins B1 and B2 display H1K activity that is characteristic of MPF• exogenously added B1 and B2 cyclins are both substrates of this kinase activity• kinase activity associated with cyclins B1 and B2 oscilates with the same kinetics as p34 kinase activity

The picture squares up:

Things are not that simple in higher eukaryotes:

PARADIGM„cell cycle as a cdc2 cycle“

appealing for its simplicity

further studies have complicated the issue

SEEMED

BUT

1991 – new human p34 protein kinase (CDK2 – cell division kinase) identified by complementation of a cdc28 mutation

in S. cerevisiae(Elledge, Spottswood, EMBO J.)

CDK2 has 66% amino acid sequence identity with the CDC2Hs identified by Lee and Nurse (1987) by complementation of a cdc2 mutation in S. pombe

FIRST

• Large number of existing p34cdc2-related kinases (referred to as CDK – cyclin dependent kinases) • Large variety of existing cyclins • Certain cyclins and CDKs interact with multiple partners

• Existence of other molecules that interact with cyclin/CDK complexes

Enormous complexity of cell cycle regulating machinery thanks to:

Current status (in words)

Current status (simplified in graphics)

Aspect no. 2 – Important phenomena

Uncontrolled hyperproliferation

And the ways of approaching their cell cycle-related

molecular basis

Restricted proliferation

Proliferative diseases(cancer, psoriasis, rheumatoid arthritis, …)

Failure to replenish diseased/injured

cell type or tissue

Phenomenon:

Phenotype:

Profit: Finding biomarkersDesigning drugs

Induction of „stem cell“

proliferation

Understanding of the molecular mechanisms that regulate cell proliferation is being converted into designing novel „smart“ diagnostical and therapeutical

strategies.

A simple logic of looking at cell cycle regulators:

Positive Negative Regulators of cell cycle:

Members: CDKs, Cyclins Inhibitors of CDKs

Down-regulated & Inactivated

Up-regulated& Activated

Up-regulated & Activated

Down-regulated & Inactivated

In hyperproliferating cells:

In nonproliferating cells:

Possible causes of abnormal function of cell cycle regulators:

Abnormality intrinsic to the regulator

Deregulation of a pathway upstream to the regulator

• Mutation in the coding sequence

• Mutation in the regulatory sequence (element)

• Epigenetic change to the coding sequence

• Epigenetic change to the regulatory sequence

• Abnormal postranslational modification

• Other

Example of the complexity of upstream regulatory pathways.

The ways to address the abnormality to the regulator itself:(comparisons between normal and abnormal cells)

At the level of gene structure:• Sequence analysis (sequencing, RFLP, …)

At the level of gene expression:• Levels of mRNA (Northern blot, RT-PCR, QRT-PCR, chip analysis, hybridization in situ)

At the level of protein:• Total amount of the regulator (Western blot, immunodetection in situ)• Association of the regulator with its partners (immunoprecipitation)• Activity (in vitro determination of kinase activity)• Other aspects of the metabolism such as turnover (metabolic labelling)

cyclin E in nuclei of cells of breast carcinoma

Sophisticated ways of experimentally addressing the significance/function of cell cycle regulators:

Genetic modification• Ectopic expression - transgenesis• Increase of expression• Expression of mutant form (e.g. dominant negative mutants)• Gene „knockout“ and „knockin“ by homologous recombination• Gene „knockdown“ using siRNA strategy• Expression of tagged proteins allowing for in vivo tracking• Other

In cells cultured in vitro In animals (mouse, Drosophila, …)

Analysis of the phenotype at multiple levels

What did we learn until now?

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Important take home lessons:

Protein Function Defect(s) Tumour type(s)

p16 Cdk4/6 inhibitor Deletion ~95% T-cell ALL

Point mutation ~75% Familial melanomas

Silencing ~75% Pancreatic carcinomas

~52% Oesophageous SCC

12 - 30% NSC lung carcinomas

~30% Gliomas

~20% Bladder carcinomas

Sporadic melanomas

~95% Tumour cell lines

p18 Cdk6 inhibitor Deletion Cell ALL

Point mutation Breast carcinoma cell lines

p21 Cdk inhibitor Underexpression Cell lines which lack p53

p27 Cdk2 inhibitor Degradation Breast carcinomas

Colorectal carcinomas

cdk1 Catalytic subunit Overexpression Breast carcinomas

Prostate carcinomas

Colorectal carcinomas

Gastric carcinomas

Various defects to cell cycle regulators occur in human malignancies:(adopted from: Webster, K.R., Exp. Opin. Invest. Drugs, 1998)

Protein Function Defect(s) Tumour type(s)

cdk2 Catalytic subunit Overexpression Colorectal carcinomas

cdk4 Catalytic subunit Amplification Sarcomas, gliomas

Point mutation Familial melanoma

Overexpression Familial adenomatous polyposis

Cyclin A Activator of cdk1/2 Stabilisation Hepatocellular carcinoma

Cyclin B1 Activator of cdk1 Overexpression 88% Colorectal carcinomas

Cyclin D1 Activator of cdk4/6 Amplification 40 - 80% Breast carcinomas

Overexpression ~70% Familial adenomatous polyposis

50% B-cell lymphomas

~47% NSC lung carcinomas

~35% Head and neck SCC

25 - 50% Oesophagus SCC

~25% Bladder carcinoma

Cyclin E Activator of cdk2 Amplification ~90% Colorectal carcinomas

Overexpression 30 - 80% Breast carcinomas

~70% Prostate carcinomas

~18% Ovarian carcinomas

Gastric carcinomas

1) Most of the canonical Cdk-cyclin complexes are dispensable for cell proliferation due to a high level of functional redundancy, promiscuity, and compensatory mechanisms.

2) The specific molecular players that drive the cell cycle in mammals seem to be cell-type-specific, and new, non-canonical functions of cyclins and Cdks have been revealed. Cdk2 - the most remarkable example

Cdk2 and its activators the E-type cyclins have been long considered

essential regulators of the mammalian somatic cell cycle.

2003 - Depletion of Cdk2 (antisense oligonucleotides, siRNA) has a negligible effect on the proliferation of colon cancer cell lines. (Tetsu and McCormick, Cancer Cell). 2003 - Cdk2 null mice are fully viable and, with the exception of a dramatic meiotic failure, show no sign of developmental abnormalities indicating that this kinase is truly dispensable for the majority, if not all, of somatic cells. (Ortega et al., Nat.Genet.; Berthet et al., Curr. Biol.)

Prognostic value of expression of cell cycle regulators is not completely understood

p27 is not a good predictor for outcome

of carcinoma of the bladder !

p27 – inhibitor of CDKs

Is it a good predictor in bladder cancer ?

p27

Growth of mouse embryonal carcinoma cell lines differing in the amount of p27

p27

A.1 1.3

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EC lines A.1 X.1 1.1 1.3 1.5

MAPK

What should we keep in mind?

• functioning of cell cycle regulating molecules is not fully understood yet

• some of the generally accepted concepts of cell cycle regulation need to be revised

• new and exciting questions need to be investigated

Thank you for your attention

Questions and comments at:ahampl@med.muni.cz