Vishal Premdev Sharma

Prolonged cell cycle response of HeLa cells to low-level alkylation exposure

Alkylation chemotherapy has been a long-standing treatment protocol for human neoplasia. N-methyl-N-nitro-N-nitrosoguanidine (MNNG) is a direct-acting monofunctional alkylator. Temozolomide is a clinical chemotherapeutic equivalent requiring metabolic breakdown to the alkylating agent. Both chemicals have similar mechanistic efficacy against DNA mismatch repair-proficient tumor cells that lack expression of methylguanine methyltransferase. Clinically relevant concentrations of both agents affect replicating cells only after the first cell cycle. This phenomenon has been attributed to replication fork arrest at unrepaired O(6)-methyldeoxyguanine lesions mispaired with thymine during the first replication cycle. Here, we show, by several different approaches, that MNNG-treated tumor cells do not arrest within the second cell cycle. Instead, the population slowly traverses through mitosis without cytokinesis into a third cell cycle. The peak of both ssDNA and dsDNA breaks occurs at the height of the long mitotic phase. The majority of the population emerges from mitosis as multinucleated cells that subsequently undergo cell death. However, a very small proportion of cells, <1:45,000, survive to form new colonies. Taken together, these results indicate that multinucleation within the third cell cycle, rather than replication fork arrest within the second cell cycle, is the primary trigger for cell death. Importantly, multinucleation and cell death are consistently avoided by a small percentage of the population that continues to divide. This information should prove clinically relevant for the future design of enhanced cancer chemotherapeutics.

Circadian properties of cancer stem cells in glioma cell cultures and tumorspheres.

Increased cancer risk is linked to disruption of circadian rhythms. Cancer stem cells (CSCs) are a known cause of cancer aggressiveness, but their circadian properties have not been described. We discovered circadian rhythms in gene expression within C6 glioma tumorspheres enriched in CSCs and found that the circadian clock is particularly robust in medium lacking any growth factors. A method is introduced for identifying individual CSCs in culture for single-cell analysis. CSCs in monolayer cell culture failed to show a circadian rhythm in nuclear localization of mPER2 protein, suggesting that cell interactions or the tumor-like microenvironment within tumorspheres enable circadian timing.

Major differences between tumor and normal human cell fates after exposure to chemotherapeutic monofunctional alkylator.

The major dilemma of cancer chemotherapy has always been a double-edged sword, producing resistance in tumor cells and life-threatening destruction of nontumorigenic tissue. Glioblastoma is the most common form of primary brain tumor, with median survival at 14 months after surgery, radiation and temozolomide (monofunctional alkylator) therapy. Treatment failure is most often due to temozolomide-resistant tumor growth. The underlying basis for development of tumor cell resistance to temozolomide instead of death is not understood. Our current results demonstrate that both cervical carcinoma (HeLa MR) and glioblastoma (U251) tumor cells exposed to an equivalent chemotherapeutic concentration of a monofunctional alkylator undergo multiple cell cycles, maintenance of metabolic activity, and a prolonged time to death that involves accumulation of Apoptosis Inducing Factor (AIF) within the nucleus. A minority of the tumor cell population undergoes senescence, with minimal caspase cleavage. Surviving tumor cells are comprised of a very small subpopulation of individual cells that eventually resume proliferation, out of which resistant cells emerge. In contrast, normal human cells (MCF12A) exposed to a monofunctional alkylator undergo an immediate decrease in metabolic activity and subsequent senescence. A minority of the normal cell population undergoes cell death by the caspase cleavage pathway. All cytotoxic events occur within the first cell cycle in nontumorigenic cells. In summation, we have demonstrated that two different highly malignant tumor cell lines slowly undergo very altered cellular and temporal responses to chemotherapeutic monofunctional alkylation, as compared to rapid responses of normal cells. In the clinic, this produces resistance and growth of tumor cells, cytotoxicity of normal cells, and death of the patient.

The circadian clock modulates anti-cancer properties of curcumin

BackgroundCurcuminoids of the spice turmeric and their enhanced derivatives have much potential as cancer treatments. They act on a wide variety of biological pathways, including those regulating cell division and circadian rhythms. It is known that circadian clocks can modify cancer therapy effectiveness, according to studies aimed at optimizing treatments based on the circadian cycle. It is therefore important to determine whether treatments with curcumin or similar chemotherapeutic agents are regulated by circadian timing. Similarly, it is important to characterize any effects of curcumin on timing abilities of the circadian clocks within cancer cells.MethodsWe examined the circadian clock’s impact on the timing of cell death and cell division in curcumin-treated C6 rat glioma cells through continuous video microscopy for several days. To evaluate its persistence and distribution in cancer cells, curcumin was localized within cell compartments by imaging its autofluorescence. Finally, HPLC and spectroscopy were used to determine the relative stabilities of the curcumin congeners demethoxycurcumin and bisdemethoxycurcumin that are present in turmeric.ResultsCircadian rhythms in cell death were observed in response to low (5xa0μM) curcumin, reaching a peak several hours before the peak in rhythmic expression of mPER2 protein, a major circadian clock component. These results revealed a sensitive phase of the circadian cycle that could be effectively targeted in patient therapies based on curcumin or its analogs. Curcumin fluorescence was observed in cell compartments at least 24xa0h after treatment, and the two congeners displayed greater stability than curcumin in cell culture medium.ConclusionsWe propose a mechanism whereby curcuminoids act in a sustained manner, over several days, despite their tendency to degrade rapidly in blood and other aqueous media. During cancer therapy, curcumin or its analogs should be delivered to tumor cells at the optimal phase for highest efficacy after identifying the circadian phase of the cancer cells. We confirmed the greater stability of the curcumin congeners, suggesting that they may produce sustained toxicity in cancer cells and should be considered for use in patient care.

Abstract 4030: Celsee™ SingleCell Chip can retrieve rare single cells with 100% efficiency for genetic analysis

Tumors are comprised of heterogeneous cells. Only a specific or rare population of tumor cells is capable of metastasizing through blood. While mapping out the genome of cancer cells, gene expression variations indicative of more aggressive cancer cells might be missed among the noise. Therefore, single cell gene expression and mutation analysis provide a more comprehensive and most precise information about the heterogeneity of cancer progression and metastasis. Although there has been a surge in technologies used for selection and analysis of single cells, single cell analysis of rare cells such as circulating tumor cells (CTCs) is fraught with technical challenges. Among many used techniques to isolate single cells, the most common cell separation method is cell sorting by flow cytometry, which requires cell labeling. The numerous steps involved in cell labelling and centrifugation result in significant cell loss and changes in gene expression. In addition, centrifugation may also cause shear-induced gene expression changes in viable cells. For other single cell analysis platforms, there is a minimum input of cells ranging from 200 - 1000 cells. Rare cells like CTCs are found in the range of 1-10 per mL of blood. Another challenge is that the input volume required for these systems is very low usually in microliters. Here, we introduce a novel microfluidic chip with 250,000 microwells which captures cells at 100% efficiency down to one cell with a dynamic range over 5 logs. The underlying principle of this technology is that the geometric pattern of the microfluidic chip captures a single cell from an initial volume of up to 1 mL of fluid due to cell settling. The unique microwell structure promotes easy fluid exchange without perturbing the cells from their captured locations, thereby eliminating cell loss during multistep downstream processing. Preliminary tests have shown that single or cluster of spiked cancer cells in blood can be immunostained, both live or fixed and can also be retrieved very efficiently from this chip using a micro capillary. The retrieved single cells can further be used for several single cell genetic analysis. We will present data describing cancer gene expression and gene mutation analysis in isolated single cells. Citation Format: Kalyan Handique, Priya Gogoi, William Chow, Kyle Gleason, Auston Payne, Vishal Premdev Sharma, Yixin Wang. Celsee™ SingleCell Chip can retrieve rare single cells with 100% efficiency for genetic analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4030. doi:10.1158/1538-7445.AM2017-4030

Abstract 858: The circadian clock of glioma cells undergoing epithelial-mesenchymal transition

Highly invasive gliomas contain many cells that have changed to an enhanced migratory state through an epithelial-mesenchymal transition (EMT). These cells have the phenotype of glioma stem cells (GSCs). Similar to GSCs, glioma cells undergoing EMT show phenotypic heterogeneity, altered gene expression, and resistance to anticancer drugs along with increased invasiveness. Circadian rhythms in tumor cells influence the progression and severity of cancer and appear to regulate cell division cycles. Increased cancer incidence and progression have often been linked to disruption or deregulation of the molecular mechanism of the circadian clock. One of the major clock proteins, PER2, has been shown to play a regulatory role as a tumor and EMT suppressor in metastatic breast cancer cell lines. GSCs in tumorsphere cultures contain circadian clocks that may regulate their cancer properties. The possibility of a regulatory role for circadian rhythms in EMT was examined here. We used a standard method to induce EMT in the C6 rat glioma cell line that has known circadian rhythms in gene expression generated by the circadian clocks within its cells. EMT was induced by exchanging the cell culture medium with a serum-free stem cell medium (SCM) containing growth factors (EGF, FGF2, PDGF-alpha-beta). Cell cultures were imaged continuously with a microscope and digital camera in a cell incubator to monitor cell shape, cell death, migration, and apoptosis. During EMT, cells changed from an extended flat state to rounded and spindle shapes, ceased proliferating, and expressed EMT markers ZEB1 and vimentin. At the end of two days in SCM, 33.4% of the cells were ZEB1-positive and only 1.26% were GFAP-positive (n=3 cultures). Cell diameters after EMT were within the size range of C6 GSCs described as Hoechst-negative cells positive for stem cell markers nestin and CD133. Following EMT, small tumorspheres began to form. After initiating EMT, the rounded cells were counted at hourly intervals for up to four days after the medium exchange. As the number of post-EMT cells increased, the population size oscillated, and when examined by Lomb-Scargle periodogram analysis, four cultures had a significant period within the circadian range, 19-29 hours, (average 22.20 ±2.45 SD, p Citation Format: Arpan De, Dilshan Harshajith Beligala, Vishal Premdev Sharma, Benjamin Ryan Fry, Michael Eric Geusz. The circadian clock of glioma cells undergoing epithelial-mesenchymal transition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 858. doi:10.1158/1538-7445.AM2017-858

Abstract 268: Circadian rhythms of glioma stem cells and progenitor cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnThe circadian timing system generates and maintains daily rhythms in physiological processes throughout the body. Disruption of circadian timing is associated with higher cancer incidence and more malignant cancers, although the interactions between cancer and circadian rhythms remain unclear. Embryonic stem cells appear to lack endogenous circadian rhythms until they differentiate. We asked whether cancer stem cells (CSCs) also lack persistent circadian oscillations at the molecular level. An inability to generate circadian rhythms may be linked to the undifferentiated state of CSCs, and treatments that can induce clock gene expression might help to initiate rhythms and promote differentiation. Because CSCs appear to evade standard cancer therapies that are effective against non-CSCs new methods to force their differentiation would be useful. To examine the circadian properties of CSCs, we selected the C6 rat glioma cell line because it expresses a strong circadian rhythm in gene expression. C6 CSCs have been characterized through flow cytometry by the side-population cell sorting method that relies on the ability of CSCs to efflux the fluorescent dye Hoechst 33342. We developed a Hoechst dye-exclusion method to identify putative CSCs in C6 monolayer cultures. This cell subpopulation failed to show a circadian rhythm in nuclear transport of a fusion protein of GFP and the circadian clock component mPER2, while the remaining cells were rhythmic. Evidence that these non-rhythmic cells are CSCs was shown by their proliferation in a medium that promotes stem cell growth and their prevalence in neurospheres grown from the C6 line that are masses of CSCs and partially differentiated cells including progenitor cells. The spheres showed immunostaining for the stem cell surface marker CD-133 and they could be induced to form astrocyte and neuron-like cells according to the cell-specific markers GFAP and beta III-tubulin, respectively. To determine whether the neurospheres are capable of generating circadian rhythms, C6 cells expressing a firefly luciferase and mPER2 fusion protein under control by the mPer2 gene promoter were imaged after they formed neurospheres. Circadian rhythms in bioluminescence persisted when the cells were synchronized with a 2-hour pulse of forskolin that induces clock genes but not when the spheres were imaged without forskolin treatment. We interpret these results as indicating a lack of circadian clocks in CSCs but circadian clocks functioning in progenitor cells of neurospheres that require synchronization for coordinated circadian activity. Because CSCs in the microenvironment of tumors may also be arrhythmic, efforts to manipulate circadian clock genes should be examined when treating gliomas and possibly other cancers.nnCitation Format: Vishal P. Sharma, Michael E. Geusz. Circadian rhythms of glioma stem cells and progenitor cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 268. doi:10.1158/1538-7445.AM2013-268