Cancer cell cycle/growth/metabolism Flashcards
Masui and Markert (1971)
They conducted research on frog oocytes that were arrested in G2, only beginning M phase after progesterone stimulation. It was found that these oocytes could be induced to enter M phase through microinjection of cytoplasm from oocytes that had been stimulated by progesterone. It was determined that this effect was not caused by progesterone itself as injected progesterone did not promote maturation and that a cytoplasmic factor was sufficient to trigger a change in the phase of the cell cycle. This result was confirmed by others in the same year, including Smith and Ecker.
Timothy Hunt in 1982
Studied the cell cycle of sea urchin (Arbacia) embryos. Used 35S-methionine labelling (radiolabelled amino acid) of newly synthesized protein and SDS-PAGE electrophoresis and autoradiography of proteins isolated from these eggs. Identified the synchronous appearance and disappearance of two proteins coordinated by M phase; they terms these cyclins A and B
Lee Hartwell 1970
He set out looking at these temperature-sensitive mutants which could grow at room temp but not at 36 degrees. Used time-lapse photomicroscopy to detect temperature-sensitive budding yeast (S.cerevisae) mutants that are defective in gene functions needed at specific stages of the cell division cycle. This resulting in him and his colleagues identifying over 100 genes specifically involved in cell cycle control - so called CDC (cell division cycle) genes. One of these genes, designated CDC28 by Hartwell controls the first step in the progression through G1 phase of the cell cycle and was also called START. This gene was interesting as it was required twice for each cell cycle - for M phase and for a point in G1 known as START.
Nurse and Thuriax 1980
It was already known that a temperature-sensitive LOF in Cdc2 (Cdc28 homologue) gives rise to cycle arrest. They found a different mutation in the same gene can give rise to a small size phenotype as it was discovered to advance the timing of mitosis. This was discovered when Nurse was doing an isolation procedure on the yeast. He devised a procedure by which he centrifuged cell population to separate large elongated cells (he was looking for these as these were the ones that had arrested in the cycle + did not divide) froom normal ones. Found a group of cells that was smaller than normal - suggested they progressed more rapidly through the cell cycle. These were termed wee mutants to reflect their Scottish origin (UoE). Having made this connection, they deliberately set out to look for wee mutants. They isolated 50 new mutants with the same phenotype and found that all byt one lay on the previously identified wee1 gene. This exception was a dominant mutation of cdc2. Previously identified recessive mutations kept the cells from dividing. This meant that different mutations in the same gene could either prevent or accelerate division, further suggesting cdc2 was a critical regulator of cell division. Genetic analysis suggested a simple model - the activity of cdc2 was required to enter mitosis and wee1 was a dosage-dependent inhibitor of cdc2.
Lee and Nurse 1987
Used cloning by functional complementation to identify a human cDNA that was capable of complementing a cdc-2 defect in yeast. This suggested that the human cDNA may encode a human protein which performed the same function biologically as cdc2 in yeast. This is now know as CDK1 in humans
Pardee 1974
- Pardee measured if any given cell (hamster fibroblast) had gone through DNA replication by the incorporation of radiolabelled thymidine, measured using a spectrometer
- Removal of serum/critical AAs cell withdrew from cell cycle
- If they re-added the serum/mitogens back cell re-entered cell cycle but took long time to enter S phase (DNA rep)
- If took serum away after ~12h of having serum present cells still retract back to G1 arrest
- But if left serum for ~14/15h cell can enter S phase
- Concluded that during this interval, something is happening – cell has made decision to start but hasn’t actually started DNA rep
Zetterberg and Larsson (1985)
Discovered that, in all stages of the cell cycle, serum deprivation results in inhibition of protein synthesis. Only in post-mitotic cells (i.e. cells in the early, the first 3-4 hours of G1) did serum withdrawal force cells into quiesence. They did this using cinematographic analysis of individual 3T3 cells exposed to serum free medium. Therefore, during early G1, the cells are highly dependent on the continuous presence of serum GFs and a high level of protein synthesis. This lead to the prediction of the existence of a protein that had minimum threshold for S phase - this was D-cyclin - the function of which was predicted before it was actually defined
Landis et al 2006
Showed that cyclin D1 kinase activity was essential for the oncogene ErbB2 to initiate breast cancer development. They generate knockin mice that expressed cyclin D1 that was not able to activate the kinase activity of CDK4/6. This resulted in resistance to breast cancers initated by ErbB2-MMTV.
Choi et al 2012
Showed - That, not only are D-cyclins capable of driving mammary tumour development, but the continued expression of D-cyclins is required for tumour maintenance They used a Cre-Lox system to create tamoxifen-inducible condition cyclin D1-KO mice. These mice also had ErbB2-sriven mammary carcinomas. The effect of removing cyclin D1 from these tumuors is quite spectacular. Not only do the tumours fail to grow any further, they start to express senescence-asosciated beta-galactosidase activity which is characteristic of senescent cells Limitations - (1) Taking cyclin D1 out of whole organism, rather than just the tumour cells. It is well established that the germinal KO animals often activate compensatory mechanisms, whereas shutdown of a protein in an adult mammal may have much more profound consequences - therefore, it is good they conducted their experiments on adult animals but they still conducted KOs from the whole animal, rather than just the tumour. However, D1 heterozygouos mice showed no physiological abnormalities, suggesting D1 was largely dispensible for normal physiology of adult animals. (2) Everything was conducted on a short timescale - would be interesting to see if any durable respons occurs they were also able to show that ablation of cyclin D3 in mice bearing Notch1-driven T cell acute lymphoblastic leukaemia (T-ALL) triggered tumour cell apoptosis o They generated condition cyclin D3 knockout mice (using a similar method to the one described above) o Switched off cyclin D3 expression after the animals had already developed tumours this resulted in a drastic reduction in number of tumour cells + extension of the animal’s survival o They determined that this occurred as a result of apoptosis by testing GFP+ leukemic cells from peripheral blood, as well as tumour cells which had infiltrated spleens, with Annexin V/7-AAD (apoptosis detection kit) and analysed using flow cytometry o Therefore, cyclin D-kinase activity represents a highly selective anti-cancer strategy that specifically targets cancer cells without significantly affecting normal tissues Linitation - does not necessarily mean it is a driver mutation
Michowski et al 2020
Focussed on ESCs (bc they are known to have high CDK1 activity). Generate knockin mice expression analogue-sensitive version of CDK1 in the place of WT CDK1. These allowed identification of CDK1 substrates in essentially any organ/cell type at any stage of development. (1) they labelled substrates + performed subcellular fractionation and found a large number of substrates in the chromatin faction - suggested CDK1 substrates in ESCs were DNA bound (confirmed using ChIP seq). (2) found CDK1 has a role in epigenetic regulation - they ectopically expressed epigenetic regulators with analogue-sensitive CDK2 in vivo. Added the modified ATP to driectly label substrates with thiophosphate. Epiegentic regulators were immunoprecipitated and immunoblots were probed with an anti-thiophosphate ester antibody. This identified 18/19 tested epigentic regulators as direct Cdk1 and Cdk2 substrates. Therefore uncovered an unexpected function of CDK1 in regulating the global epigenetic landscape of PSCs Cav - Did not prove this is linked to cancer and was in mouse ES/iPSCs
Fojer er al 2005
found that, in conditions of serum starvation, triple knockout (TKO) mouse embryonic fibroblasts of all three pocket proteins, were able to pass through the R checkpoint, but were subsequently arrested at G2. This suggested that loss of pocket protein function is insufficient to allow cell cycle progression and other mutations, that permit evasion of G2 and apoptosis, are also needed for tumorigenesis
Walter et al 2019
generated a genetically engineered mouse model of lung cancer that enabled Cre-dependent inactivation and subsequent activation of RB in KRAS-driven lung adenocarcinomas. Surprisingly, reactivation of RB did little to slow the proliferation of cancer cells, but instead caused the cancer cells to differentiate towards a less metastatic phenotype. This suggests that RB loss may play an important role in metastasis and it is suggested that reactivation of RB may be a promising therapeutic for patients with lung adenocarcinoma
Serrano et al 1997
One of the mutant alleles of HRAS is G12V Transduced human, mouse embryonic and rat embryonic fibroblasts with a retrovirus encoding HRAS G12V or a control virus. They observed that introducing G12V abolished the increase in cell number that we see in the control culture - i.e. the cells stopped proliferating. They then used a propidium iodide-BrdU flow cytometry method to determine DNA content per cell as well as newly synthesized DNA in the fibroblasts. It was seen that there was a significant decrease in the S phase fraction following introduction of the mutant RAS allele. Even on day 1 after tarnsductoin we can see a decrease frim 31% ti 5.4% and by 3 days we can see the S phase fraction is almost completely gone. Therefore, mutant RAS in these cells led to cell cycle arrest, rather than proliferation How this was working at a molecular level. They did Western blotting (without a loading control - caveat - could have had unequal amounts of protein loaded) of lysates from fibroblasts transudced with retrovirus encoding HRAS G12V or control retrovirus. They found a pretty dramatic increase in p53 levels in all three types of fibroblasts following G12V transfection. C
Harrington
Dual key model for oncogene induced proliferation
Winters et al 1998
investigated influence of p53 on radiation-induced G2 cell cycle arrest using H1299 cells expressing temperature-sensitive p53 o H1299 cells = a human non-small cell lung carcinoma cell line derived from the lymph node, which is widely used in research. As with other immortalised cell lines, H1299 cells can divide indefinitely. They cells have a homozygous partial deletion of the TP53 gene and, as a result, do not express the tumour suppressor p53 protein which in part accounts for their proliferative propensity o Gamma-irradiated cells lacking p53 arrested transiently in G2 with Cdc2 extensively phosphorylated at inhibitory sites and both Cdc2 and cyclin B1 restricted to the cytoplasm o P53 activated by temperature shift results in more protracted (lasting for a longer time than expected) G2 arrest that could not be overridden by checkpoint-abrogating drugs o Surprisingly, this enhancement of G2 arrest was associated with a marked lack of inhibitory phosphorylation of Cdc2 and with the nuclear localisation of both Cdc2 and cyclin B1 o Concluded that a p53-dependent pathway can operate after exposure of human cells to ionising radiation to promote G2 arrest accompanied by nuclear translocation rather than inhibitory phosphorylation of Cdc2
Rieder et al 1995
it was found via video microscopy that laser ablation of the last unattached kinetochore accelerated mitotic exit of Ptk1cells6, leading to the suggestion that even a single unattached kinetochore is able to generate a signal capable of delaying anaphase.
Hanks et al 2004
studied patients with mosaic variegated aneuploidy (MVA), a rare condition characterised by mosaic aneuploidies in a range of different chromosomes and tissues, with the proportion of aneuploid cells more than 25% of that of unaffected individuals. The risk of malignancy in these patients is high, with rhabdomyosarcoma, Wilms tumour or leukaemia usually occurring before two years of age. This group screened the gene encoding BubR1 (BUB1B) by conformation-sensitive gel electrophoresis in 8 families with MVA and found that 5 of these had biallelic mutations in the gene; each family had one missense mutation and another mutation resulting in protein truncation or an absent transcript. These combinations of the two different mutations could have been due to chance, but also could be because biallelic truncating mutations may result in more extensive aneuploidy and subsequent embryonic lethality, whereas biallelic missense mutations may not change BubR1 function enough to cause MVA. Interestingly, two of the five patients developed rhabdomyosarcoma in later life, suggesting cancer predisposition14.
Rao et al 2005
showed that ApcMin/+ mice, carrying one mutant Apc allele developed ~0.4 colon tumours per mouse by 3 months, but in contrast ApcMin/+ mice that also had decreased BubR1 levels (BubR1+/-) developed 10-fold more tumours by the same age16. Therefore, BubR1 may function as a driver of tumorigenesis in the presence of other tumorigenic mutations, such as ApcMin/+. Again this could say the extent to which…
Jiao et al 2021
found upregulation of BubR1 in CCA cell lines and human tissues and interestingly showed that, via a subcutaneous xenograft tumour model, that BubR1 knockouts were able to suppress tumorigenicity, with reductions in tumour weight and volume in comparisons to control notes. To explore further this role in tumorigenicity, the group found via protein-microarray analysis that the knockout of BubR1 suppressed expression of phosphorylation c-Jun, furthering this by showing that Jun and JNK proteins were increased in CCA tissue samples via immunohistochemistry staining. The group suggest that these findings indicate BubR1 may mediate tumour progression via the JNK-c-Jun signalling pathway20, which may be distinct from its role in the SAC.
Mulcahy 1985
they microinjected the protein products of the RAS oncogene into NIH 3T3 cells. Following this, the recipient cells were able to initiate DNA synthesis in the absence of added serum. They further showed that NIH 3T3 cells that were induced to divide through addition of serum, were not able to enter S phase following microinjection of anti-RAS antibodies. This demonstrated a requirement of RAS for S phase transition
Filmus et al 1994
a relationship was seen between activated RAS and overexpression of cyclin D1 in epithelial cells from the rat intestine. o In their study, they transfected intestinal cells with an inducible RAS expression vector. o They treated these with anti-sense cyclin D1 oligonucleotides and this led to a reduction in rate of cell proliferation. o This led to the conclusion that RAS-induced cyclin D1 contributed to the higher rate of proliferation caused by the RAS oncogene in intestinal epithelial cells “
Hartmann in 1928
Prevented a growth amoeba in G2 phase from entering mitosis indefinitely by periodically resecting a portion of its cytoplasm, thereby preventing the attainment of a presumed critical size
Johnston, Pringle and Hartwell 1977
Looked at S.cerevisae. Used temperature-sensitive cell division cycle mutations again which limited cell division/the cell cycle. They saw that these cells were still able to grow (increased volume, mass and protein content). Then the stopped the cells from growing (nitrogen starvation) and observed that cells that had completed their cycles arrested in G1. Concluded that a specific event early in G1 could not be completed until a critical size is attained
