Anticancer drugs affect temperature signaling and epigenetic factors in the cold tolerance of Caenorhabditis elegans

doi:10.21203/rs.3.rs-2261648/v1

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Anticancer drugs affect temperature signaling and epigenetic factors in the cold tolerance of Caenorhabditis elegans

https://doi.org/10.21203/rs.3.rs-2261648/v1

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Drugs are vital for maintaining the body healthy and treating diseases. As most drugs have side effects, it is important to gain a complete understanding of their action mechanisms. However, significant cost and time are involved in elucidating their mechanisms. We conducted drug screening at a low cost and in a short time using the phenomenon of cold tolerance in the nematode Caenorhabditis elegans. Among ~ 4000 drugs, we screened the anticancer drugs leptomycin B and camptothecin that affect cold tolerance. Leptomycin B and camptothecin inhibited molecular pathway(s) downstream of the thermosensory signaling via the cGMP-dependent channel TAX-4 in ASJ thermosensory neurons and the thermoreceptor DEG-1 in ASG thermosensory neurons. Leptomycin B affected cold tolerance by inhibiting the molecular pathway upstream of the insulin receptor DAF-2 that regulates cold tolerance in the intestine. Camptothecin decreased the expression levels of genes required for epigenetic processes, such as hrde-1 and deps-1 encoding Argonaute and constitutive P granule protein, respectively. Moreover, hrde-1 and deps-1 mutants exhibited abnormal cold tolerance. This study established an experimental model for drug screening using the cold tolerance of C. elegans and proposed that an anticancer drug upregulates cold tolerance via temperature signaling and epigenetic regulation.

Biological sciences/Genetics

Biological sciences/Neuroscience

Caenorhabditis elegans

Drug screening

Cold tolerance

Camptothecin

Leptomycin B

Drugs are essential for maintaining our health; however, their side effects are sometimes harmful. Therefore, a precise understanding of their mechanism of action and side effects is necessary for their safe use in the clinical setting. However, screening new drugs and elucidating their mechanism of action involve significant cost and time. Fission yeast and the nematode Caenorhabditis elegans have been used as suitable genetic model animals for drug screening and investigating the mechanism of action. Leptomycin A and B produced by Streptomyces sp. have been identified as substances inducing cell elongation of the fission yeast via screening of antifungal antibiotics ¹. CRM1 has been isolated by genetic analysis using a fission yeast mutant that is resistant to leptomycin B (LMB) and exhibits abnormal deforming of nuclear chromosome domains under restrictive temperatures ^2,3. LMB inhibits nuclear export of proteins with a leucine-rich nuclear export signal (NES), such as Rev proteins, by functioning as an inhibitor of CRM1/exportin1 directly binding to the NES ^4–7. Genome-wide RNAi screening in C. elegans has suggested that silencing xpo-1, cording exportin1 in humans, enhances autophagy by increasing the activity of the nuclear HLH-30/TFEB transcription factor that is involved in the transcriptional modulation of autophagy ⁸ .

The anticancer drug camptothecin (CPT) was isolated from the tree Camptotheca acuminata in China. An analysis of cultured cells indicated that CPT inhibits DNA synthesis by acting as an inhibitor of topoisomerase I ⁹. During DNA replication, topoisomerase I forms a complex with DNA by a covalent bond. CPT inhibits DNA replication by interacting with the topoisomerase I–DNA complex ^{9 10}. Although the budding yeast and fission yeast are sensitive to CPT, in the yeast mutant with top1 deletion, CPT exerted no influence on cording topoisomerase I ^11–13. Genetic analysis of yeast using CPT has resulted in the isolation of various genes involved in DNA replication and repair. Saccharomyces cerevisiae with a mutation in RAD52, which is involved in DNA repair, was hypersensitive to CPT ^12,14. The budding yeast mutant lacking serine 129 in histone 2A (H2A ser129) exhibited a hypersensitive phenotype to CPT. H2A ser129 is a crucial component that efficiently repairs DNA double-stranded breaks¹⁵. Yeasts with mutations in Tof1 and Csm3, which function as checkpoint-mediator proteins during DNA replication, were also found to be hypersensitive to CPT ¹⁶. Although yeast tof1Δ exhibited hypersensitive to CPT, this abnormality of tof1Δ is suppressed by Sir1-4 mutation. Inhibition of deacetylation of histone H4-K16 by the inactivation of SIR protein promotes CPT resistance ¹⁷. Hence, fission yeast and cultured animal cells have been used as an experimental system for drug screening and investigation of their target molecules and mechanism of action. However, it is difficult to examine how a drug affects other cells and tissues using yeast or cultured cells because they can only reveal the mechanism of action in a single cell.

The nematode C. elegans is a multicellular model animal that can be used with ease for genetic screening and epistasis analysis. The adult C. elegans body consists of approximately 1000 somatic cells and a genome containing approximately 20,000 genes, which is approximately similar to the gene number in humans. Although C. elegans is a very simple animal, it has numerous homologous genes and common mechanisms conserved between C. elegans and humans. C. elegans has basic tissues such as the nervous system, intestines, gonads, and muscles. An analysis of numerous isolated C. elegans mutants enables the analysis of neural circuits and tissue networks.

It has been reported that C. elegans can be used as a model animal of diseases for drug screening and elucidation of disease pathogenesis. For instance, a number of mutants defective in genes involved in Parkinson’s disease have been isolated in C. elegans. A phenotypic analysis of these mutants for Parkinson’s disease model might help identify the pathogenic mechanism of Parkinson’s disease ¹⁸. A C. elegans TDP-43 model, a genetic model of ALS, is suitable for the phenotypic screening of candidate therapeutic compounds, among which the neuroleptic pimozide that blocks T-type Ca²⁺ channels has been identified as a promising drug for ALS treatment ^19,20.

Our previous studies suggested that cold tolerance and cold acclimation in C. elegans can be used as an experimental system to elucidate neural circuits and tissue networks ^21,22. Cold tolerance and cold acclimation are regulated by the temperature received through three head sensory neurons, ASJ, ADL, and ASG. The temperature information received by ASJ opens the cGMP-dependent channel TAX-4 through the G protein pathway and promotes insulin secretion. The insulin secreted by ASJ is received by the insulin receptor DAF-2 in the intestine, and FOXO-type transcription factors control cold tolerance by regulating the expression of cold tolerance-related genes ²¹. Through an unidentified thermoreceptor, ADL receives temperature information that opens the TRPV channels OCR-1, OCR-2, and OSM-9 ^23,24. Moreover, OCR-2, and OSM-9 regulate cold acclimation by functioning as a thermoreceptor in ADL ²⁵. The DEG/ENaC-type mechanoreceptor DEG-1, also known as a mechanosensory neuron, receives information on not only mechanical stimuli but also temperature from ASG. The information on temperature stimuli received from ASG is transmitted to the downstream interneurons AVH and AIN via XDH-1 encoding xanthine dehydrogenase ^26,27. Thus, multiple novel genes could be identified and their molecular mechanisms, neural circuits, and tissue networks could be elucidated by investigating cold tolerance and cold acclimation.

In this study, we identified through screening of ~ 4000 drugs that LMB and CPT could affect cold tolerance and cold acclimation in C. elegans. We also elucidated the genetic epistasis between LMB or CPT and the genes involved in cold tolerance or cold acclimation by evaluating various mutants cultivated in a culture plate containing medium impregnated with LMB or CPT. This experimental system could be used as an experimental model for drug screening and investigating the mechanism of action of novel drugs.

Drug screening using the experimental system of cold tolerance

We have previously reported that C. elegans exhibits cold tolerance and cold acclimation depending on the cultivation temperature. When animals are cultivated at 25°C from the egg to adult stage, they cannot survive at 2°C. However, those cultivated at 15°C can survive at 2°C ²¹. Animals can acclimate to new temperatures within a few hours, a phenomenon known as cold acclimation ^21–24. For instance, when animals cultivated at 15°C are transferred to 25°C for 3 h, they cannot survive at 2°C by replacing the previous cultivated temperature with the new experienced temperature in only a few hours at individual level.

We hypothesized that cold tolerance and cold acclimation can be used as an experimental model for drug screening and investigation of the action mechanism of drugs. Therefore, we established an experimental system using the phenomenon of cold tolerance in wild-type C. elegans strains exposed to various medicinal substances. The wild-type strains were cultivated at 22.5°C in a nematode growth medium (NGM) impregnated with approximately 4000 medicinal substances (Fig. 1a and b). After reaching the adult stage, they were rapidly exposed to a low-temperature stimulus of 3°C (Fig. 1a, c). Although wild-type strains cultivated at 22.5°C could not survive at 3°C, those grown in NGM impregnated with several types of medicinal substances exhibited abnormal increment of cold acclimation. As a first screening, we isolated medicinal substances that increased the survival rate of wild-type strains by 10% (Fig. 1b). Among 4000 compounds, animals cultivated on NGM with CPT, LMB, and dihydrolotenone showed increment of cold tolerance, in first screening. Because these three compounds induced larval arrest (Fig. 1c), 22.5°C-grown animals were cultivated to young adults on NGM without the chemicals and then transferred to new NGM with chemicals, in second screening. After 22.5°C-grown animals were exposed to 3°C, CPT and LMB-treated animals increased the survival rate by 20% (Fig. 1b), but animals exposed to Dihydrolotenone did not increase the survival rate. We therefore continued to examine the effects of CPT and LMB on cold tolerance.

Cpt And Lmb Affect Cold Tolerance And Cold Acclimation

It has been reported that CPT inhibits DNA replication by interacting with the DNA–topoisomerase I complex^9,10, and LMB inhibits the nuclear export of proteins by functioning as an inhibitor of CRM1/expotin1, which encodes a nuclear transporter^4–7. A structural analysis of leptomycins indicated that they belong to the unsaturated, branched-chain fatty acids with 3-lactone rings at the end¹(Fig. 1c). In the present study, the C. elegans strains cultivated in NGM impregnated with CPT and LMB failed to hatch from eggs or were arrested at the L4 larval stage (Fig. 1c). Therefore, we cultivated animals from the egg to L4 larval stage at the desired temperature, transferred them into NGM impregnated with CPT and LMB, and then returned them to the previous cultivated temperature for approximately 24 h before exposure to cold stimuli (Fig. 2a and b). We first tested the concentrations at which LMB and CPT affect the cold tolerance phenotype. Wild-type animals cultivated at 25°C were exposed to 0, 0.1, 1, and 10 µM LMB and CPT, respectively, and then subjected to a cold stimulus of 2°C. Wild-type animals cultivated at 25°Ccould not survive at 2°C (Fig. 2c [0 µM] and Fig. 2d [0 µM]). However, the wild-type strains exposed to 1 and 10 µM CPT and 10 µM LMB exhibited a significant increase in cold tolerance (Fig. 2c [1 and 10 µM] and Fig. 2d [10 µM]). These findings suggest that at least 1 and 10 µM concentrations of CPT and LMB, respectively, affect the cold tolerance of C. elegans.

To investigate the effects of CPT and LMB on cultivation temperature–dependent cold stimuli, we cultivated wild-type strains under exposure to 10 µM of CPT and LMB at various temperatures and evaluated their cold tolerance and cold acclimation. When wild-type control animals were cultivated at 15°C without exposure to CPT and LMB, almost animals could survive at 2°C for 2 h (Fig. 2e and f). Similarly, wild-type animals exposed to CPT and LMB exhibited almost 100% survival rates, respectively (Fig. 2e and f). To examine the effect on cold tolerance in animals cultivated at 15°C, we applied the cold stimulus of 2°C for a longer period of 96 h, wherein the survival rates of wild-type animals decreased to ~ 23%. However, wild-type animals exposed to CPT demonstrated a higher survival rate than wild-type control animals (Fig. 2g). Interestingly, wild-type animals exposed to LMB showed a decrease of cold tolerance that was in contrast to the phenotype of wild-type animals cultivated at 22.5°C in the drug screening (Fig. 2h). These results suggest that CPT increases cold tolerance and LMB decreases cold tolerance under the cultivation condition of 15°C.

We next conducted a cold tolerance test on wild-type control animals exposed to CPT and LMB. When cultivated at 20°C or 25°C and then exposed to 2°C, these animals exhibited a survival rate of approximately 5% or 25%, respectively (Fig. 3a–d). However, wild-type animals exposed to CPT exhibited a significant increase in cold tolerance when cultivated at 25°C (Fig. 3c). These results indicate that CPT increases cold tolerance at 25°C but exerts no effect on the phenotype of cold tolerance at 20°C. Similarly, in wild-type strains cultivated at 20°C or 25°C, LMB significantly increased their cold tolerance at both temperatures (Fig. 3b and d). These findings suggest that LMB increases cold tolerance in wild-type animals cultivated at 20°C and 25°C but decreases it in the animals cultivated at 15°C.

To explore the effect of CPT and LMB in animals acclimated at 25°C for 4 h, we evaluated their cold tolerance after cultivation from the egg to adult stage at 15°C. When wild-type control animals cultivated at 15°C were transferred to 25°C for 4 h, ~ 90% of them were acclimated to 25°C and killed by the 2°C cold stimulus (Fig. 3e and f). Wild-type animals exposed to CPT more rapidly acclimated to 25°C for 4 h than the wild-type control animals (Fig. 3e), indicating that CPT increases cold acclimation in this protocol. LMB significantly increased the survival rate of animals acclimated to 25°C for 4 h from 15°C (Fig. 3f, Wild-type with LMB), whose phenotype was similar to that of animals cultivated at 20°C and 25°C (Fig. 3b, d, Wild-type with LMB).

Cpt And Lmb Affect Temperature Signaling Pathways In Asg

To determine the impact of chemical substances on tissues, we examined the drug effect in various mutants defective in genes that regulate cold tolerance or cold acclimation. XDH-1 encoding xanthine dehydrogenase controls cold tolerance by regulating the response to temperature in AIN and AVH^26,27. Although wild-type animals cultivated at 15°C could survive at 2°C for 48 h, xdh-1 mutant control animals could not survive at 2°C (Fig. 2e and f). To examine genetic epistasis between xdh-1 and the molecular pathway that is inhibited by the chemicals, we evaluated cold tolerance in xdh-1 mutant exposed to CPT or LMB. We observed that xdh-1 mutant exposed to CPT exhibited a 15% higher survival rate than the xdh-1 mutant control (Fig. 2e). Moreover, xdh-1 mutant exposed to LMB exhibited a significant increase in cold tolerance (Fig. 2f). These results indicate that the abnormal decrease of cold tolerance in xdh-1 mutant was suppressed by some genes inhibited by CPT and LMB, suggesting that XDH-1 regulates cold tolerance upstream of a molecular pathway inhibited by CPT and LMB under the 15°C cultivating condition.

The DEG/ENaC-type mechanoreceptor DEG-1 regulates cold tolerance by acting as a thermoreceptor in the sensory neuron ASG, which is located upstream of the interneurons AIN and AVH ²⁶. When animals cultivated at 15°C were exposed to 2°C for 96 h, the deg-1 mutant control exhibited a significant decrease in cold tolerance compared with the wild-type control (Fig. 2g and h). deg-1 mutant exposed to CPT showed an increase in survival rate that was almost similar to that of wild-type animals exposed to CPT (Fig. 2g). These results suggest that DEG-1 in ASG regulates cold tolerance upstream of the genes inhibited by CPT. LMB did not affect the phenotype of cold tolerance in the deg-1 mutant, although it decreased cold tolerance in the wild-type control (Fig. 2h). These data imply that LMB could affect the same pathway involved in cold tolerance as that by DEG-1 in ASG.

Some Genes Inhibited By Cpt And Lmb Affect Cold Tolerance Regulation In Asj

The head sensory neuron ASJ receives temperature information and transmits it via TAX-4 encoding the cGMP-dependent channel ²¹. When animals were cultivated at 20°C, the tax-4 mutant control showed a significantly higher survival rate than the wild-type control (Fig. 3a and b). The abnormal cold tolerance of tax-4 mutant was decreased by CPT treatment (Fig. 3a), suggesting that TAX-4 in ASJ is epistatic to a molecular pathway inhibited by CPT at 20°C. Treatment with LMB enhanced the abnormal increase of cold tolerance in the tax-4 mutant control, whose phenotype was significantly higher than that of wild-type animals treated with LMB (Fig. 3b). These results suggest that a molecular pathway inhibited by LMB is downstream or parallel with the tax-4-mediated pathway that regulates temperature signaling in ASJ under cold tolerance.

The thermosensory neuron ASJ receives temperature information and secretes insulin, which is received by the insulin receptor DAF-2 in the intestine ²¹. The daf-2 mutant control exhibited an abnormal increase in cold acclimation when cultivated at 25°C (Fig. 3c and d). daf-2 mutant exposed to CPT showed a similar survival rate as that of the daf-2 mutant control, although wild-type animals exposed to CPT exhibited a significant increase in cold acclimation compared with the wild-type control animals (Fig. 3c). This result indicates that genes inhibited by CPT are involved in cold tolerance in at least the same pathway of the DAF-2 signaling pathway. The cold tolerance of daf-2 mutant exposed to LMB was significantly decreased compared with the wild-type control whose phenotype was almost similar to that of daf-2 control (Fig. 3d). These findings suggested that a molecular pathway inhibited by LMB is upstream for the daf-2-mediated pathway involved in cold tolerance at 25°C.

CPT and LMB affect cold acclimation regulated by TRPV channels in ADL and DAF-2 in the intestine

As reported earlier, the TRPV channels OSM-9, OCR-2, and OCR-1 control cold tolerance and cold acclimation by regulating the response to temperature in ADL ^23–25. When animals cultivated at 15°C were transferred to 25°C for 4 h, the osm-9 ocr-2; ocr-1 mutant exhibited an abnormal increase in cold acclimation compared with the wild-type control (Fig. 3e and f). The osm-9 ocr-2; ocr-1 triple mutant exposed to CPT showed a significant increase in cold acclimation compared with the wild-type animals exposed to CPT, whose phenotype was similar to that of the osm-9 ocr-2; ocr-1 mutant control (Fig. 3e). These findings suggested that CPT inhibits genes regulating temperature acclimation in upstream of OSM-9, OCR-2 and OCR-1. Although LMB increased temperature acclimation of wild-type, LMB did not affect the temperature acclimation of osm-9 ocr-2; ocr-1 triple mutant (Fig. 3f). These results suggest that genes inhibited by LMB could act in the same pathway of the TRPV channels OSM-9, OCR-2, and OCR-1 that regulate cold acclimation in the thermosensory neuron ADL.

Exploration Of Downstream Molecules Affected By Lmb And Ctp

To explore the downstream molecules affected by CTP and LMB, we performed RNA sequencing analysis to screen for genes whose expression levels were altered by the chemicals. Because 10 µM of CPT and LMB increased the cold acclimation of animals cultivated at 25°C (Fig. 2c and d), we extracted RNA from wild-type strains cultivated at 25°C without exposure to chemicals and from wild-type strains cultivated on plates treated with chemicals and compared the expression levels of all genes in C. elegans (Figs. 4, 5, 6a). Animals exposed to CPT exhibited a significantly increased expression of genes involved in stress response, proteolysis general, extracellular general, lysosome, and metabolism (Fig. 6b) and a decreased expression of genes involved in extracellular material, metabolism, transcription: chromatin and ribosome (Fig. 6c). Animals exposed to LMB showed a significantly increased expression of genes related to extracellular material, stress response, proteolysis general, and metabolism (Fig. 6d) and a decreased expression of genes related to stress response, ribosome, extracellular material, mRNA functions, cell cycle, DNA, metabolism, proteolysis general, lysosome, and chaperone (Fig. 6e).

We evaluated the cold tolerance and cold acclimation of mutants defective in genes whose expression levels were altered according to the chemicals. The expression of numr-1 encoding a nematode-specific protein was significantly increased by treatment with LMB (Fig. 5). When wild-type strains were cultivated at 25°C and transferred to 15°C for 3 h, they exhibited an approximately 60% survival rate. However, approximately 90% of numr-1 mutants could not survive at 2°C (Fig. 6f). These results suggest that NUMR-1 regulates cold acclimation by functioning downstream of LMB.

hrde-1 encoding Argonaute (Ago) and deps-1 encoding the constitutive P granule protein were downregulated by CPT exposure (Fig. 4). In C. elegans, HRDE-1 has been reported to be involved in epigenetics by associating with endogenously expressed siRNAs that induce gene silencing ²⁸. DEPS-1, a key component of P granules, regulates efficient gene silencing through the piRNA pathway ²⁹. Wild-type animals cultivated at 20°C exhibited a survival rate of approximately 20%, whereas half of hrde-1 mutants could survive at 2°C. These results suggest that the decreased expression of hrde-1 and deps-1 caused by CPT stress in wild-type animals could affect the genes regulating cold tolerance, causing a decreased cold tolerance phenotype. We hypothesized that genes involved in epigenetic processes were involved in cold tolerance. We evaluated the cold tolerance of mutants defective in Argonaute protein RDE-1 (RNA interference-deficient 1) and DNA demethylase NMAD-1, respectively. Interestingly, both rde-1 mutant and nmad-1 mutant exhibited an abnormal increase in cold tolerance (Fig. 6g). These findings suggested that genes related to epigenetic processes are involved in cold tolerance.

Screening drugs and elucidating their mechanism of action are both expensive and time-consuming. In this study, we established an experimental system of screening for drugs involved in temperature–dependent phenomena at a low cost and in a short time using the cold tolerance and cold acclimation phenotypes of C. elegans. We have previously isolated various genes regulating cold tolerance and cold acclimation and proposed their intracellular mechanisms or intertissue networks^21–26. In the present study, we elucidated the genetic relationship between the pathway inhibited by the drugs LMB and CPT and the genes involved in the temperature response of various tissues in cold tolerance and cold acclimation by conducting an epistasis analysis on drugs and various mutants. In animals cultivated at 15°C, the mutants of the DEG/ENaC-type mechanoreceptor DEG-1 and xanthine dehydrogenase XDH-1 exhibited an abnormal decrease in cold tolerance. In the sensory neuron ASG, DEG-1 receives temperature information and transmits it to the interneurons AVH and AIN, wherein XDH-1 acts as a modulator of the neuronal activity of AVH and AIN^26,27. Under 15°C cultivation condition, the epistasis analysis indicated that CPT affects cold tolerance downstream of DEG-1 and XDH-1 (Fig. 7a), and that LMB affects cold tolerance downstream of XDH-1(Fig. 7a). CPT and LMB function to increase the survival rate of xdh-1 mutants under cold stimuli, whereas xdh-1 mutants treated with CPT and LMB showed a significantly lower cold tolerance than that of wild-type animals treated with CPT and LMB (Fig. 2e, f). These data imply that CPT and LMB could also affect cold tolerance through a parallel pathway with XDH-1 (Fig. 7a). Treatment with LMB did not improve the decrease in the cold tolerance of deg-1 mutants, although LMB decreased the cold tolerance of wild-type animals (Fig. 2h). These findings suggest that genes inhibited by LMB regulate cold tolerance at least in the same pathway as that of DEG-1, but the epistatic relationship between the pathway inhibited by LMB and DEG-1 has not been determined (Fig. 7a).

Animals cultivated at 20°C receive temperature information and transduce the signal in the thermosensory neuron ASJ through the cGMP-dependent channel TAX-4²¹. The results of the epistatic analysis on tax-4 mutants and the chemical substances suggested that CPT and LMB affect cold tolerance downstream of TAX-4 at 20°C (Fig. 3a and b). Treatment with CPT decreased the survival rate of tax-4 mutants under cold stimuli, whereas tax-4 mutant treated with CPT exhibited higher cold tolerance than the wild-type animals treated with CPT (Fig. 3a). Treatment with LMB enhanced the abnormal increase of cold tolerance in tax-4 mutants, whereas tax-4 mutant treated with LMB exhibited a higher survival rate than the wild-type strain treated with LMB (Fig. 3b). These results imply that treatment with CPT and LMB could affect cold tolerance by inhibiting the pathway not only downstream of TAX-4 but also in a separate pathway (Fig. 7b).

The insulin receptor DAF-2 regulates temperature acclimation in the intestine [²¹]. Although CPT and LMB increased the cold tolerance of wild-type animals, they did not affect temperature acclimation in the daf-2 mutant (Fig. 3c and d). However, wild-type animals exposed to CPT exhibited a similar cold tolerance to that of daf-2 mutant (Fig. 3c), suggesting that CPT affects cold tolerance at least in the same pathway as that of DAF-2 in the intestine, but the epistatic relationship between the molecular pathway inhibited by CPT and DAF-2 has not been clarified (Fig. 7c). In daf-2 mutant treated with LMB, daf-2 mutation suppressed the increase of cold tolerance in wild-type animals treated with LMB, which showed a similar phenotype to that of daf-2 control (Fig. 3d). These genetic analyses indicated that LMB affected temperature acclimation upstream of DAF-2, which regulates cold tolerance in the intestine (Fig. 7c).

Animals cultivated at 15°C can acclimate to 25°C for only a few hours^21,24,25,30. The TRPV channels OSM-9, OCR-2, and OCR-1 regulate temperature acclimation in the thermosensory neuron ADL^23–25. CPT and LMB increased temperature acclimation in wild-type animals, but they did not affect the abnormal temperature acclimation in the osm-9 ocr-2; ocr-1 triple mutant (Fig. 3e and f). This result suggests that CPT regulates temperature acclimation upstream of OSM-9, OCR-2, and OCR-1. There was no significant difference in temperature acclimation between the wild-type animals treated with LMB and the osm-9 ocr-2; ocr-1 mutants treated with LMB (Fig. 3f). These results suggest that OSM-9, OCR-2, and OCR-1 regulate temperature acclimation through the same pathway as that of the genes inhibited by LMB, but we did not determine the epistatic relationship between the target genes of LMB and OSM-9, OCR-2, OCR-1 involved in temperature acclimation.

The genetic epistatic analyses using cold tolerance and temperature acclimation revealed the genetic relationship between the drug and tissues such as neurons and the intestine. Our results suggested that the drugs affect cold tolerance and temperature acclimation not only in the same pathway but also in parallel pathways with several genes. CPT and LMB could inhibit the activity of various genes regulating cold tolerance and temperature acclimation. LMB inhibits nuclear transfer proteins and possibly affects cold tolerance and temperature acclimation by significantly altering the expression of genes involved in extracellular material, stress responses, proteolysis general, metabolism, ribosome, and mRNA function (Fig. 6d and e [**P < 0.01]). Wild-type animals treated with CPT showed changes in the expression level of genes involved in stress response, proteolysis general, and extracellular matrix (Fig. 6b and c [**P < 0.01]). It is possible that CPT, which acts as a topoisomerase I inhibitor, would affect cold tolerance and temperature acclimation by altering the expression level of these genes. Treatment with CPT decreased the expression levels of hrde-1 encoding Argonaute (Ago) and deps-1 encoding the constitutive P granule protein. Mutants defective in epigenetic factors exhibited an abnormal increase in cold tolerance. These results imply that anticancer drugs involved in DNA replication and repair such as CPT alter temperature responses by affecting the expression of genes involved in epigenetic regulation (Fig. 7d).

We established an experimental system of genetic analysis for drug screening and elucidation of the mechanism of action using the phenomena of cold tolerance and temperature acclimation in the nematode C. elegans. Using this experimental system, we can investigate the epistatic relationship between unknown drugs and genes and their effects on tissues in an inexpensive and efficient manner.

Ethical issues and approval

All animal treatments in this research were performed in accordance with the Japanese Act on Welfare and Management of Animals (Act No. 105 of October 1, 1973; latest revisions Act No. 51 of June 2, 2017, Effective June 1, 2018). All experimental protocols were approved by the Institutional Animal Care and Use Committees of Konan University.

Strains

The standard procedures for culturing and handling C. elegans were essentially described by Brenner ³¹. We used the following C. elegans strains: N2 Bristol England; KHR067 xdh-1(ok3234); TU38 deg-1 (u38); FK127 tax-4(p678); CB1370 daf-2(e1370); FG125 ocr-2(ak47) osm-9(ky10); ocr-1(ak46); VC2552 nmad-1(ok3133); hrde-1(tm1200); DG3226 deps-1(bn124); WM27 rde-1(ne219); and RB1749 numr-1(ok2239) III.

Cold Tolerance Test For Drug Screening

A chemical library for the drug screening was prepared from Takeda Pharmaceutical Company. Culture plates were prepared for drug screening as follows: 150 µl of Nematode Growth Medium (NGM) ³¹ was added to 96-well plates per well. Enriched Escherichia coli OP50 and compounds were added to the NGM agar to a final concentration of 40 µM chemicals and dried for ~ 4–8 h. To collect sufficient number of eggs for evaluating cold tolerance, several adult animals (P0) were placed onto the NGM plate in 224 x 224mm square dishes and incubated for ~ 6 days, until F1 animals reached the adult stage and the food source became low. The F1 adult animals were collected by washing S-basal buffer ³². The collected F1 animals were dissolved in 3.5 ml egg-isolation solution (hypochlorous acid 1mL + water 2.15 mL + 10N NaOH 0.35mL). Adult carcasses were removed by repeated centrifugation and washing. The solution containing only F2 eggs was transferred to 96-wells plates containing NGM and compounds, so that each well contained ~ 30–60 eggs. The animals were cultivated at 22.5°C for ~ 2–3 days until they grew from the egg to adult stage. The plates were placed on ice for 15 min and transferred to a freezer at 3°C for 48 h. After exposure to cold stimuli, the plates were taken out from the refrigerator, and after ~ 2–12 h, the number of live and dead animals were counted to calculate survival rates. Secondary screening for the drugs that inhibited animal growth was conducted as follows: Animals were cultured in NGM medium without chemical-addition until young adults, then were placed into a 3-cm dish containing chemical-added NGM, and maintained for 24 hours. The plates were placed on ice for 15 min and transferred to a freezer at 3°C for 48 h. After cold stimuli, the number of live and dead animals were counted to calculate survival rates.

Cold Tolerance And Cold Acclimation Assay

Cold tolerance and cold acclimation tests were conducted as described previously²¹. Animals were cultivated in 2% (w/v) agar NGM plates containing Escherichia coli OP50 in 3.5-cm-diameter plastic dishes. Single or several well-conditioned adults (P0) were placed at the desired temperature and incubated for 15–20 or 3–5 h, respectively, until they had laid ~ 100 eggs. P0 animals were then removed to synchronize the growth of F1 animals. The F1 animals were incubated from the egg to L4 larval or young adult stage at 15°C for ~ 120 h and 20°C for ~ 67 h. The animals were collected using M9 buffer in a 1.5-ml tube and then washed at least two times with M9 buffer. 10 mM, 1 mM or 0.1 mM chemical stock previously prepared in pure dimethyl sulfoxide (DMSO), was then added to the NGM agar to a final concentration of 10 µM, 1 µM or 0.1 µM chemical respectively with 0.1% DMSO as solvent. The NGM without chemicals for control experiment was prepared by adding DMSO to NGM to a final concentration of 0.1% DMSO. The washed animals were transferred to NGM plates containing CPT or LMB and dried up for approximately 10 min. The plates were placed in a light-shielding container. In the cold tolerance test of wild-type animals exposed to 0, 0.1, 1, and 10 µM LMB and CPT, the animals were cultivated at 20°C from egg to young adult. The animals were transferred to 25°C for 24 h after exposure to drugs. In the cold tolerance test of xdh-1(ok3234), deg-1(u38), or tax-4(p678), the animals were cultivated at the desired temperature for ~ 24 h after exposure to drugs. In the cold tolerance test for cultivation at 25°C, daf-2(e1379) animals were cultivated at 15°C from the egg to L4 larval or young adult stage, because daf-2(e1379) undergoes dauer arrest at 25°C. daf-2(e1370) strains cultivated at 15°C were transferred to 25°C for 24 h after exposure to drugs. In the cold acclimation test of ocr-2(ak47) osm-9(ky10); ocr-1(ak46), the animals were cultivated at 15°C for ~ 20 h after exposure to drugs, and then transferred to 15°C for 4 h. After cultivation at each temperature, the plates were immediately chilled on ice for 20 min and then transferred to a 2°C refrigerator (CRB-41A, Hitachi, Japan) for 2, 24, 48, or 96 h. After exposure to cold stimuli, the assay plates were incubated at 15°C until the nematodes started moving. Survival rates were calculated by counting the number of live and dead animals. Each cold tolerance and cold acclimation test was conducted using more than three plates per day. The number of live and dead animals was counted on plates containing more than 30 nematodes. All data were obtained by assaying the results from at least three independent days.

Rna Sequencing Analysis

Total RNA was extracted from wild-type N2 strain treated with CPT and LMB. Animals were cultivated in 2% (w/v) agar NGM plates containing E. coli OP50 in 3.5-cm-diameter plastic dishes. Several well-conditioned adults (P0) were placed until they had laid ~ 100 eggs. P0 animals were then removed to synchronize the growth of F1 animals. The F1 animals were incubated from the egg to young adult stage at 20°C for ~ 67 h. Young adult animals were collected using M9 buffer from the cultivation plates, placed into a 1.5-ml tube, and washed twice with M9 buffer. The washed animals were transferred to NGM plates impregnated with 10 µM LMB and CPT. Control animals were placed in 3.5-cm plates without transferring to NGM supplemented with LMB and CPT. After cultivating the animals at 25°C for 20 h, they were collected and washed with M9 buffer (22 mM KH₂PO₄, 42 mM Na₂HPO₄, 85 mM NaCl, and 1 mM MgSO₄) twice. The Maxwell RSC Simply RNA tissue kit (Promega; AS1340) with Maxwell RSC Instrument (Promega; AS4500) was used to extract total RNA according to the manufacturer’s instructions. Sequencing libraries were prepared using a TruSeq Stranded mRNA Sample Prep Kit (Illumina) according to the manufacturer’s instructions. Libraries were quantified using a Bioanalyzer (Agilent) and then sequenced on an Illumina NovaSeq 6000 instrument to a depth of at least 13.3 mega read 60 million reads using 150-bp paired end reads. RNA-seq data have been deposited in the DRA of DDBJ with the accession number DRA015100. Raw RNA-seq reads were analyzed using the CLC Genomics Workbench 21 (Qiagen, Germany).

An online tool for annotation and visualization, WormCat was used for gene set enrichment analysis (Fig. 6b-e) ³³. We categorized gene sets whose expression were significantly up-regulated (FDR < 0.01, logFC > 0.6) or down-regulated (FDR < 0.01, logFC <- 0.6) by treatment of CPT or LMB. The gene set was mapped to the C. elegans “Whole genome v2”, following the standard WormCat Flow analysis parameters.

Statistical analysis

All error bars in the figures indicate SEM.

Assuming that all data follow normal distribution, all statistical analyses were conducted using parametric tests, i.e., the Tukey–Kramer method. Multiple comparisons were performed using one-way analysis of variance, with comparisons tested using the Tukey–Kramer method at *P < 0.05 and **P < 0.01. The tests were performed using Mac statistical analysis version 3 (Esumi, Japan). All graphs were prepared using GraphPad Prism 9 (GraphPad Software, USA).

Data Availability

RNA-seq data have been deposited and released in the DDBJ BioProject with the accession number PRJDB14678 (https://ddbj.nig.ac.jp/resource/bioproject/PRJDB14678). The datasets generated during in this study are available from the corresponding author on reasonable request.

Acknowledgments

We thank the National Bioresource Project (Japan) and the Caenorhabditis Genetic Center for strains; T. Ujisawa, M. Fujita and Takeda Pharmaceutical Company Limited for supporting experiments and discussion. A.K. and A.O. were supported by the Kinoshita Memorial Foundation, Naito Foundation, Takeda Science Foundation, Suzuken Memorial Foundation, Hirao Taro Foundation of Konan Gakuen for Academic Research, and the Asahi Glass Foundation, Mochida Memorial Foundation for Medical and Pharmaceutical Research, Shimadzu Science and Technology Foundation, A.K. was supported by the AMED PRIME (22gm6510004h0002), and JSPS KAKENHI (22H05512, 21H02534, 21K19279, and 22H05512). A.O. and M.O. were supported by JSPS KAKENHI(20J30004 & 21K15141[M.O.] and 21K06275 [A.O.]). M.O. was supported by Kawanishi Memorial ShinMaywa Education Foundation.

Author contributions

M.O., K.N., A.O., and A.K. designed the research; M.O., N.S., K.N., A.O., and A.K. performed the research; M.O., N.S., K.N., A.O., and A.K. analyzed data; and M.O., N.S., K.N., A.O., and A.K. wrote the paper.

The authors declare no competing interest.

Competing interests

The authors declare no competing interests.

Hamamoto, T., Gunji, S., Tsuji, H. & Beppu, T. Leptomycins A and B, new antifungal antibiotics. I. Taxonomy of the producing strain and their fermentation, purification and characterization. J Antibiot (Tokyo). 36, 639–645 (1983).
Adachi, Y. & Yanagida, M. Higher order chromosome structure is affected by cold-sensitive mutations in a Schizosaccharomyces pombe gene crm1 + which encodes a 115-kD protein preferentially localized in the nucleus and its periphery. J Cell Biol. 108, 1195–1207 (1989).
Nishi, K. et al. Leptomycin B targets a regulatory cascade of crm1, a fission yeast nuclear protein, involved in control of higher order chromosome structure and gene expression. J Biol Chem. 269, 6320–6324 (1994).
Wolff, B., Sanglier, J. J. & Wang, Y. Leptomycin B is an inhibitor of nuclear export: inhibition of nucleo-cytoplasmic translocation of the human immunodeficiency virus type 1 (HIV-1) Rev protein and Rev-dependent mRNA. Chem Biol. 4, 139–147 (1997).
Stade, K., Ford, C. S., Guthrie, C. & Weis, K. Exportin 1 (Crm1p) is an essential nuclear export factor. Cell. 90, 1041–1050 (1997).
Fornerod, M., Ohno, M., Yoshida, M. & Mattaj, I. W. CRM1 is an export receptor for leucine-rich nuclear export signals. Cell. 90, 1051–1060 (1997).
Fukuda, M. et al. CRM1 is responsible for intracellular transport mediated by the nuclear export signal. Nature. 390, 308–311 (1997).
Silvestrini, M. J. et al. Nuclear Export Inhibition Enhances HLH-30/TFEB Activity, Autophagy, and Lifespan. Cell Rep. 23, 1915–1921 (2018).
Hsiang, Y. H., Hertzberg, R., Hecht, S. & Liu, L. F. Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. J Biol Chem. 260, 14873–14878 (1985).
Hertzberg, R. P. et al. Irreversible trapping of the DNA-topoisomerase I covalent complex. Affinity labeling of the camptothecin binding site. J Biol Chem. 265, 19287–19295 (1990).
Bjornsti, M. A., Benedetti, P., Viglianti, G. A. & Wang, J. C. Expression of human DNA topoisomerase I in yeast cells lacking yeast DNA topoisomerase I: restoration of sensitivity of the cells to the antitumor drug camptothecin. Cancer Res. 49, 6318–6323 (1989).
Eng, W. K., Faucette, L., Johnson, R. K. & Sternglanz, R. Evidence that DNA topoisomerase I is necessary for the cytotoxic effects of camptothecin. Mol Pharmacol. 34, 755–760 (1988).
Nitiss, J. & Wang, J. C. DNA topoisomerase-targeting antitumor drugs can be studied in yeast. Proc Natl Acad Sci U S A. 85, 7501–7505 (1988).
Knab, A. M., Fertala, J. & Bjornsti, M. A. Mechanisms of camptothecin resistance in yeast DNA topoisomerase I mutants. J Biol Chem. 268, 22322–22330 (1993).
Redon, C. et al. Yeast histone 2A serine 129 is essential for the efficient repair of checkpoint-blind DNA damage. EMBO Rep. 4, 678–684 (2003).
Redon, C., Pilch, D. R. & Bonner, W. M. Genetic analysis of Saccharomyces cerevisiae H2A serine 129 mutant suggests a functional relationship between H2A and the sister-chromatid cohesion partners Csm3-Tof1 for the repair of topoisomerase I-induced DNA damage. Genetics. 172, 67–76 (2006).
Puddu, F. et al. Chromatin determinants impart camptothecin sensitivity. EMBO Rep. 18, 1000–1012 (2017).
Cooper, J. F. & Van Raamsdonk, J. M. Modeling Parkinson's Disease in C. elegans. J Parkinsons Dis. 8, 17–32 (2018).
Patten, S. A. et al. Neuroleptics as therapeutic compounds stabilizing neuromuscular transmission in amyotrophic lateral sclerosis. JCI Insight. 2 (2017).
Giunti, S., Andersen, N., Rayes, D. & De Rosa, M. J. Drug discovery: Insights from the invertebrate Caenorhabditis elegans. Pharmacol Res Perspect. 9, e00721 (2021).
Ohta, A., Ujisawa, T., Sonoda, S. & Kuhara, A. Light and pheromone-sensing neurons regulates cold habituation through insulin signalling in Caenorhabditis elegans. Nat Commun. 5, 4412 (2014).
Sonoda, S., Ohta, A., Maruo, A., Ujisawa, T. & Kuhara, A. Sperm Affects Head Sensory Neuron in Temperature Tolerance of Caenorhabditis elegans. Cell Rep. 16, 56–65 (2016).
Ujisawa, T. et al. Endoribonuclease ENDU-2 regulates multiple traits including cold tolerance via cell autonomous and nonautonomous controls in Caenorhabditis elegans. Proc Natl Acad Sci U S A. 115, 8823–8828 (2018).
Okahata, M., Wei, A. D., Ohta, A. & Kuhara, A. Cold acclimation via the KQT-2 potassium channel is modulated by oxygen in Caenorhabditis elegans. Sci Adv. 5, eaav3631 (2019).
Ohnishi, K. et al. OSM-9 and OCR-2 TRPV channels are accessorial warm receptors in Caenorhabditis elegans temperature acclimatisation. Sci Rep. 10, 18566 (2020).
Takagaki, N. et al. The mechanoreceptor DEG-1 regulates cold tolerance in Caenorhabditis elegans. EMBO Rep. 21, e48671 (2020).
Okahata, M., Motomura, H., Ohta, A. & Kuhara, A. Molecular physiology regulating cold tolerance and acclimation of Caenorhabditis elegans. Proc Jpn Acad Ser B Phys Biol Sci. 98, 126–139 (2022).
Buckley, B. A. et al. A nuclear Argonaute promotes multigenerational epigenetic inheritance and germline immortality. Nature. 489, 447–451 (2012).
Suen, K. M. et al. DEPS-1 is required for piRNA-dependent silencing and PIWI condensate organisation in Caenorhabditis elegans. Nat Commun. 11, 4242 (2020).
Okahata, M. et al. Natural variations of cold tolerance and temperature acclimation in Caenorhabditis elegans. J Comp Physiol B. 186, 985–998 (2016).
Brenner, S. The genetics of Caenorhabditis elegans. Genetics. 77, 71–94 (1974).
Lewis, J. A. & Fleming, J. T. Basic culture methods. Methods Cell Biol. 48, 3–29 (1995).
Holdorf, A. D. et al. WormCat: An Online Tool for Annotation and Visualization of Caenorhabditis elegans Genome-Scale Data. Genetics. 214, 279–294 (2020).

No competing interests reported.

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Anticancer drugs affect temperature signaling and epigenetic factors in the cold tolerance of Caenorhabditis elegans

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Abstract

Figures

Introduction

Results

Drug screening using the experimental system of cold tolerance

Cpt And Lmb Affect Cold Tolerance And Cold Acclimation

Cpt And Lmb Affect Temperature Signaling Pathways In Asg

Some Genes Inhibited By Cpt And Lmb Affect Cold Tolerance Regulation In Asj

Exploration Of Downstream Molecules Affected By Lmb And Ctp

Discussion

Conclusions

Materials And Methods

Ethical issues and approval

Strains

Cold Tolerance Test For Drug Screening

Cold Tolerance And Cold Acclimation Assay

Rna Sequencing Analysis

Statistical analysis

Declarations

References

Additional Declarations

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