From research to rapid response: mass COVID-19 testing by volunteers at the Centre for Genomic Regulation.

The COVID-19 pandemic has posed and is continuously posing enormous societal and health challenges worldwide. The research community has mobilized to develop novel projects to find a cure or a vaccine, as well as to contribute to mass testing, which has been a critical measure to contain the infection in several countries. Through this article, we share our experiences and learnings as a group of volunteers at the Centre for Genomic Regulation (CRG) in Barcelona, Spain. As members of the ORFEU project, an initiative by the Government of Catalonia to achieve mass testing of people at risk and contain the epidemic in Spain, we share our motivations, challenges and the key lessons learnt, which we feel will help better prepare the global society to address similar situations in the future.

Cell death pathways in response to antitumor therapy.

Failure to eliminate cancer cells that have been exposed to cytotoxic agents may contribute to the development of resistance to antitumor drugs. A widespread model in present day oncology is that antitumor therapy involves the triggering of tumor cells to undergo apoptosis, and cells that can avoid apoptosis will be resistant to such therapy. Apoptosis is a defined program of cell death that is markedly influenced by the fact that many routes leading to it are mutated or deregulated in human cancer. Mutations in the tumor suppressor protein p53, a common feature of many cancers, may decrease the sensitivity of cells to some antitumor agents. Moreover, it has been increasingly reported that antitumor therapy not only causes apoptosis, but other forms of cell death as well, such as mitotic catastrophe, necrosis and autophagy, or a permanent cell arrest with phenotype characteristics of senescence. Mitotic catastrophe is a form of cell death that results from abnormal mitosis, which does not seem to depend on wild-type p53. Sometimes mitotic catastrophe is used restrictively for faulty mitosis leading to cell death, which may occur via apoptosis or necrosis. We critically review herein how antitumor therapy may elicit the response of human cancers through different cell pathways leading to cell death.

Mithramycin SK modulates polyploidy and cell death in colon carcinoma cells.

During a normal cell cycle, polyploidy and aneuploidy can be prevented by several checkpoints, which are mainly p53 dependent. Here, we show that treatment of HCT-116 (p53+/+) colon carcinoma cells with the novel antitumor antibiotic mithramycin SK (MSK) results in polyploidization and mitotic catastrophe, which occurs after a transient halt in G1 phase followed by the overtaking of the G2-M checkpoint when treated cells are incubated in a fresh drug-free medium. Cells reentering aberrant mitosis mainly died by necrosis, although active caspase-3 was observed. Our results indicate that a decrease in p53 RNA and protein levels, together with concomitant changes in the expression of other proteins such as p21WAF1, were involved in MSK-induced polyploidy. Furthermore, the effects of MSK on HCT-116 (p53+/+) cells cannot be attributed exclusively to the down-regulation of p53 by MSK, because these effects differed from those observed in MSK-treated HCT-116 (p53-/-) cells. The p53(-/-) cells died mainly from G2-M through early p53-independent apoptosis, which appeared to be mediated by caspase-2, although secondary necrosis was also observed.

Circumvention of the multidrug-resistance protein (MRP-1) by an antitumor drug through specific inhibition of gene transcription in breast tumor cells.

Multidrug-resistance protein 1 (MRP-1) confers resistance to a number of clinically important chemotherapeutic agents. The promoter of the mrp-1 gene contains an Sp1-binding site, which we targeted using the antitumor bis-anthracycline WP631. When MCF-7/VP breast cancer cells, which overexpress MRP-1 protein, were incubated with WP631 the expression of the multidrug-resistance protein gene decreased. Conversely, doxorubicin did not alter mrp-1 gene expression. The inhibition of gene expression was followed by a decrease in the activity of the MRP-1 protein. The IC(75) for WP631 (drug concentration required to inhibit cell growth by 75%) circumvented the drug-efflux pump, without addition of resistant modifiers. After treatment with WP631, MCF-7/VP cells were committed to die after entering mitosis (mitotic catastrophe), while treatment with doxorubicin did not affect cell growth. This is the first report on an antitumor drug molecule inhibiting the mrp-1 gene directly, rather than being simply a poor substrate for the transporter-mediated efflux. However, both situations appeared to coexist, thereby a superior cytotoxic effect was attained. Ours results suggest that WP631 offers great potential for the clinical treatment of tumors displaying a multidrug-resistance phenotype.

Mechanisms of drug-induced mitotic catastrophe in cancer cells.

Mitotic catastrophe is a mechanism of cell death characterized by the occurrence of aberrant mitosis with the formation of large cells that contain multiple nuclei, which are morphologically distinguishable from apoptotic cells. Sometimes, mitotic catastrophe is used restrictively to indicate a type of cell death that occurs during or after a faulty mitosis leading to cell death, which takes place via necrosis or apoptosis, rather than a cell death itself. Several antitumor drugs and ionizing radiation are known to induce mitotic catastrophe, but precisely how the ensuring lethality is regulated or what signals are involved is barely characterized. The type of cell death resulting from antitumor therapy can be determined by the mechanism of action of the antitumor agent, dosing regimen of the therapy, and the genetic background in the cells being treated. Wild-type p53 promotes apoptosis or senescence, while mitotic catastrophe is independent of p53. Mitotic catastrophe can be regarded as a delayed response of p53-mutant tumors that are resistant to some damage. In this context, the elucidation of the mechanisms of treatment-induced mitotic catastrophe should contribute to an improvement of the antitumor therapy, because most of the solid tumors bear an inactive p53 protein.

Cellular response and activation of apoptosis by mithramycin SK in p21(WAF1)-deficient HCT116 human colon carcinoma cells.

HCT116 (p21(-/-)) human colon carcinoma cells treated with mithramycin SK (MSK), a novel analog of the antitumor antibiotic mithramycin A (MTA), were transiently arrested in G2/M, with some cells entering a faulty mitotic cycle without cytokinesis that resulted in G1-like cell arrest, which consisted of post-mitotic aneuploid G1 cells. Some of these cells synthesized DNA and elicited an apoptotic response. The absence of p21(WAF1) made HCT116 cells more sensitive to MSK than to the related MTA. MSK also showed higher antiproliferative activity than MTA on HCT116 cells with different genetic backgrounds, including those lacking the p53 gene. Apoptosis in MSK-treated p21(-/-) cells involved caspase 2 rather than caspase 3. Untreated HCT116 (p21(-/-)) cells presented a little caspase 3 activity, which increased slightly after treatment with MSK. The apoptotic response in p21(-/-) cells comprised caspase 2 acting as an executor caspase together with a loss of mitochondrial membrane potential that may be initiated by caspase 2. In contrast, caspase 3 was activated in wild-type HCT116 after treatment with MSK.

A nuclear budding mechanism in transiently arrested cells generates drug-sensitive and drug-resistant cells.

HCT116 (p53(+/+)) human colon carcinoma cells treated with nanomolar concentrations of doxorubicin underwent transient senescence, synthesized DNA, showed endopolyploidization, increased their size and became multinucleated without a significant increase in mitosis. Nuclei underwent a budding process that involved the release of buds outside the nuclear membrane, and some of the buds seemed to escape from the polyploid cells. A clonogenic assay showed that some cells proliferated following the initial treatment. In general, cells ensuing after budding were not resistant to a variety of drugs, although some of them turned out to be resistant, indicating a potential selective advantage. Nuclear budding was accompanied by changes in protein levels in the giant cells, including inhibition of p53 and enhanced expression of p21(WAF1) and the meiosis-related Mos. The buds might be a mechanism for the segregation and elimination of redundant DNA, or for generating viable aneuploid cells with a potentially extended life span.

Mitotic catastrophe as a consequence of chemotherapy.

According to a widespread model, anti-cancer chemotherapy involves the triggering of tumor cells to undergo apoptosis, so apoptosis-resistant cells would be recalcitrant to such therapy. However, in addition to apoptosis, which is mainly dependent on the activity of the tumor suppressor protein p53, cells can be eliminated following DNA damage by other mechanisms. Mitotic catastrophe, a form of cell death that results from abnormal mitosis, is one such mechanism. While the term mitotic catastrophe has been used to describe a type of cell death that occurs during mitosis, there is still no broadly accepted definition. Occasionally, mitotic catastrophe is used restrictively for abnormal mitosis leading to cell death, which can occur through necrosis or apoptosis, rather than cell death itself. Although different classes of cytotoxic agents induce mitotic catastrophe, the pathways of abnormal mitosis differ depending on the nature of the inducer and the status of cell-cycle checkpoints. Moreover, mitotic catastrophe can also develop because of aberrant re-entry of tumor cells into the cell cycle after prolonged growth arrest. Elucidation of the factors that regulate different aspects of treatment-induced mitotic catastrophe should assist in improving the efficacy of anti-cancer therapy, providing opportunities for the development of new drugs.

Apoptosis and cell recovery in response to oxidative stress in p53-deficient prostate carcinoma cells.

We have studied the effects of different concentrations of H(2)O(2) on the proliferation of PC-3 prostate carcinoma cells. Since this cell line lacks functional p53, we sought to characterize whether apoptotic response to the oxidative insult was altered such that, unlike in cells containing functional p53 apoptosis may be reduced and replaced by other mechanisms of cellular arrest and death. We did not observe necrosis in PC-3 cells treated with H(2)O(2) concentrations of up to 500 microM. In the presence of 50 microM H(2)O(2), arrest was observed in the G2-phase of the cell cycle, along with p53-independent apoptosis. In the presence of 500 microM H(2)O(2), addition of l-buthionine sulfoximine increased the percentage of apoptotic cell death. Senescence-associated cell arrest was never observed. Moreover, some of the treated cells seemed to be resistant to oxidative damage. These cells re-entered the cell cycle and proliferated normally. Analysis of the expression of p21(waf1) and of p21 protein levels, as well as the activity of caspase-3 and caspase-8, allowed us to characterize some aspects of the arrest of PC-3 cells in G2 and the apoptotic response to oxidative stress in the absence of functional p53.