Live Cell Imaging: Assessing the Phototoxicity of 488 and 546 nm Light and Methods to Alleviate it.

In live cell imaging of fluorescent proteins, phototoxicity of the excitation light can be problematical. Cell death is obvious, but reduced cell viability can make the interpretation of observations error prone. We characterized the phototoxic consequences of 488 and 546 nm light on untransformed human cells and tested methods that have or could be used to alleviate photodamage. Unlabeled RPE1 cells were given single 0.5-2.5 min irradiations in early G1 from a mercury arc lamp on a fluorescence microscope. Four hundred eighty-eight nanometer light produced a dose-dependent decrease in the percentage of cells that progressed to mitosis, slowing of the cell cycle for some of those entering mitosis, and a ∼12% incidence of cell death for the highest dose. For 546 nm light we found a 10-15% reduction in the percentage of cells entering mitosis, no strong dose dependency, and a ∼2% incidence of cell death for the longest irradiations. For cells expressing GFP-centrin1 or mCherry-centrin1, fewer entered mitosis for each dose than unlabeled cells. For constant total dose 488 nm light irradiations of unlabeled cells, reducing the intensity 10-fold or spreading the exposures out as a series of 10 sec pulses at 1 min intervals produced a minor and not consistent improvement in the percentage of cells entering mitosis. Reducing oxidative processes, by culturing at ∼3% oxygen or adding the reducing agent Trolox noticeably increased the fraction of cells entering mitosis. Thus, for long-term imaging there can be value to using RFP constructs and for GFP-tagged proteins reducing oxidative processes. J. Cell. Physiol. 232: 2461-2468, 2017. © 2016 Wiley Periodicals, Inc.

The SWI/SNF ATPases Are Required for Triple Negative Breast Cancer Cell Proliferation.

The Brahma (BRM) and Brahma-related Gene 1 (BRG1) ATPases are highly conserved homologs that catalyze the chromatin remodeling functions of the multi-subunit human SWI/SNF chromatin remodeling enzymes in a mutually exclusive manner. SWI/SNF enzyme subunits are mutated or missing in many cancer types, but are overexpressed without apparent mutation in other cancers. Here, we report that both BRG1 and BRM are overexpressed in most primary breast cancers independent of the tumor's receptor status. Knockdown of either ATPase in a triple negative breast cancer cell line reduced tumor formation in vivo and cell proliferation in vitro. Fewer cells in S phase and an extended cell cycle progression time were observed without any indication of apoptosis, senescence, or alterations in migration or attachment properties. Combined knockdown of BRM and BRG1 showed additive effects in the reduction of cell proliferation and time required for completion of cell cycle, suggesting that these enzymes promote cell cycle progression through independent mechanisms. Knockout of BRG1 or BRM using CRISPR/Cas9 technology resulted in the loss of viability, consistent with a requirement for both enzymes in triple negative breast cancer cells.

Link between DNA damage and centriole disengagement/reduplication in untransformed human cells.

The radiation and radiomimetic drugs used to treat human tumors damage DNA in both cancer cells and normal proliferating cells. Centrosome amplification after DNA damage is well established for transformed cell types but is sparsely reported and not fully understood in untransformed cells. We characterize centriole behavior after DNA damage in synchronized untransformed human cells. One hour treatment of S phase cells with the radiomimetic drug, Doxorubicin, prolongs G2 by at least 72 h, though 14% of the cells eventually go through mitosis in that time. By 72 h after DNA damage we observe a 52% incidence of centriole disengagement plus a 10% incidence of extra centrioles. We find that either APC/C or Plk activities can disengage centrioles after DNA damage, though they normally work in concert. All disengaged centrioles are associated with γ-tubulin and maturation markers and thus, should in principle be capable of reduplicating and organizing spindle poles. The low incidence of reduplication of disengaged centrioles during G2 is due to the p53-dependent expression of p21 and the consequent loss of Cdk2 activity. We find that 26% of the cells going through mitosis after DNA damage contain disengaged or extra centrioles. This could produce genomic instability through transient or persistent spindle multipolarity. Thus, for cancer patients the use of DNA damaging therapies raises the chances of genomic instability and evolution of transformed characteristics in proliferating normal cell populations.