Cell Cycle Assays-Part I

This is the first part of a series of blogs about using fluorescent proteins in cell based assays with established examples, a common theme here at the AlleleBlog.

FUCCI Cell Cycle Sensor

The FUCCI Cell Cycle Sensor is composed of a red (RFP) and a green (GFP) fluorescent protein fused to different regulators of the cell cycle: cdt1 and geminin.

During the cell cycle, these two proteins are ubiquitinated at different time points by specific ubiquitin E3 ligases, which tag them for degradation in the proteasome. The E3 ligases’ activities are regulated temporally and result in the biphasic cycling of GERMINI and CDT1 levels during the cell cycle. In the G1 phase of the cell cycle, GERMINI is degraded; therefore, only CDT1 tagged with RFP is present and appears as red fluorescence within the nuclei. In the S, G2, and M phases, CDT1 is degraded; only GERMINI tagged with GFP is present, resulting in cells with green fluorescent nuclei.

During the G1/S transition, when CDT1 levels are decreasing and GERMINI levels increasing, both proteins are present, so are the tagged fluorescent proteins. When the green and red images are overlaid, nuclei fluoresce yellow. This dynamic color change, from red-to-yellow-to-green, represents the entire cell cycle. This representation can be used to study the effects of elements that may influence cell cycles.

Sakaue-Sawano A, Kurokawa H, Morimura T, Hanyu A, Hama H, Osawa H, Kashiwagi S, Fukami K, Miyata T, Miyoshi H, Imamura T, Ogawa M, Masai H, Miyawaki A.Visualizing spatiotemporal dynamics of multicellular cell-cycle progression. Cell. 2008 Feb 8;132(3):487-98.

CCNB1-CyclinB(NT)-GFP

In late S phage, CCNB1 promoter will be switched on to drive the expression of Cyclin B N-terminus-GFP expression; thereafter the fluorescent signal will be switched off at the destruction box in Cyclin B N-terminus at the end of Mitosis phase. During the intervening phase the fusion reporter protein will translocate from cytoplasm to nucleus by the cytoplasmic retention signal in the Cyclin B N-terminus.

Thomas N. Lighting the circle of life: fluorescent sensors for covert surveillance of the cell cycle. Cell Cycle. 2003 Nov-Dec;2(6):545-9.

GFP-PCNA/YFP-PCNA
GFP-PCNA, a fusion of GFP and PCNA, has been widely used as a convenient tool to monitor the progress of S phase. At the onset of S phase, GFP-PCNA translocates into the nucleus; at mitosis the nuclear envelope breaks down and the nuclear accumulation of PCNA-GFP dissipates.

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lenti-shRNA expression vector with Puromycin-2A-RFP for custom RNAi virus packaging only. email: RNAi@allelebiotech.com

Promotion of the Week 102510-103110:

Promotion of the week: prepackaged lentivirus expressing IL15, IL2, $25 off, this week only.

Tags: cell based assays, cell cycle, cell cycle arrest, DNA-repair, Fluorescent proteins, M phase, mitosis, S Phase

DNA Repair Pathway Factors in Cell-Based Screening for Restoring Patients’ Sensitivity to Cancer Therapies

Cancers undergoing therapies may develop resistance to treatment. Many current cancer treatments, such as cisplatin, function by creating DNA damage, particularly to fast-dividing cells, i.e., most cancer cells. These treatments may be rendered ineffective by DNA-damage response pathways. Cancer resistance to therapies may come from increased activity in nonhomologous end joining, decreased functions of mismatch repair, or reactivation of the Fanconi anemia (FA)/BRCA DNA-damage response pathway, etc. Ironically the loss of function of some of these DNA-damage repair factors may have partially caused the cancer formation in the first place. Regaining their functions in cancer cells possibly contribute to drug resistance. Molecules that disrupt FA/BRCA pathway or other DNA-damage responses could be used to help restore therapy sensitivity.

Like many proteins that function in DNA-damage repair complexes, FANCD2, a member of the FA pathway factor group, is targeted towards chromatin following damage to DNA in a process called foci formation. There have been recent studies that monitored the foci formation of GFP-FANCD2 in small molecule library screening and identified inhibitors to FANCD2 as candidates for a cancer therapy sensitizer. The assays can be improved in a number of ways. There are fluorescent proteins (FPs) that are much brighter than EGFP for increased sensitivity. For instance, the monomeric green FP mWasabi is about 2-3 fold brighter than EGFP, with narrower emission peak, and is more stable under acidic environment. The newly developed lancelet YFP (LanYFP, developed/introduced by Allele Biotech) is astonishingly 10 times brighter than EGFP. Since it has a longer excitation and emission wavelength, it should inherently have a better signal to noise/background ratio compared to EGFP because cells autofluoresce less in long wavelengths. The improved brightness would also help in this respect. The fold difference between foci and LanYFP background will be the same as EGFP, but the contrast will still probably be better because of less autofluorescent background and significantly higher fluorescence reading in foci.

Other factors that may be used as a screening target when fused to effective FPs may probably include:

1) Homologous recombination (HR)
a. End Resection
MRN complex (MRE11, RAD50, NBS1)
CtIP, RPA, ATM, ATR, Exo1, BLM, RMI1, TopIIIa, DNA2, BRCA1
b. Synapsis
RAD51, BRCA2, PALB2, RAD51B, RAD51C, RAD51D, RAD51AP1, XRCC2, XRCC3, RAD54, RAD54B
c. DNA synthesis
DNA polymerase delta, PCNA

2) Nonhomologous End Joining (NHEJ)
Ku70/Ku80, DNA-PK, Ligase IV, XRCC4, XLF

3) Fanconi Anemia Pathway
FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL, FAAP100, FANCM, MHF, FAAP24, FANCD2, FANCI, FAN1, FANCN, FANCJ, FANCM

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Puromycin-resistant versions of lanRFP (red fluorescent protein from lancelet) for mammalian expression, just became available this week. ABP-FP-RCNCS1P, ABP-FP-RNNCS1P

Promotion of the Week 101110-101710:

30% off the brightest ever lancelet YFP, ABP-FP-YPNCS10, $349 reduced to $244.3 for this week’s orders only.

Tags: cancer therapy, cancer treatment, DNA-damage, DNA-repair, drug resistance, drug screening, fluorescent protein, NIH grant, RNAi