Efficacy of a new cancer treatment strategy based on eradication of tumor-initiating stem cells in a mouse model of Krebs-2 solid adenocarcinoma.
ABSTRACT: Krebs-2 solid carcinoma was cured using a new "3+1" strategy for eradication of Krebs-2 tumor-initiating stem cells. This strategy was based on synchronization of these cells in a treatment-sensitive phase of the cell cycle. The synchronization mechanism, subsequent destruction of Krebs-2 tumor-initiating stem cells, and cure of mice from a solid graft were found to depend on the temporal profile of the interstrand cross-link repair cycle. Also, the temporal profile of the Krebs-2 interstrand repair cycle was found to have a pronounced seasonal cyclicity at the place of experiments (Novosibirsk, Russia). As a result, the therapeutic effect that is based on application of the described strategy, originally developed for the "winter repair cycle" (November-April), is completely eliminated in the summer period (June-September). We conclude that ?ne of the possible and the likeliest reasons for our failure to observe the therapeutic effects was the seasonal cyclicity in the duration of the interstrand repair cycle, the parameter that is central to our strategy.
Project description:We describe the strategy, which allows curing experimental mice engrafted with Krebs-2 ascites. The strategy is based on the facts that i) Krebs-2 tumor-initiating stem cells (TISCs) are naturally capable of internalizing fragments of extracellular double-stranded DNA (dsDNA); ii) upon delivery into TISCs, these dsDNA fragments interfere with the on-going DNA repair process so that TISCs either die or lose their tumorigenic potential. The following 3-step regimen of therapeutic procedures leading to eradication of Krebs-2 ascites is considered. Firstly, three timed injections of cyclophosphamide (CP) exactly matching the interstrand cross-link (ICL) repair phases that lead to synchronization of ascites cells in late S/G2/M. Secondly, additional treatment of ascites 18 hours post each CP injection (at NER/HR transition timepoint) with a composite dsDNA-based preparation interfering with the NER and HR repair pathways, so that tumorigenic properties of ascites cells are compromised. Thirdly, final treatment of mice with a combination of CP and dsDNA injections as ascites cells undergo apoptotic destruction, and the surviving TAMRA+ TISCs arrested in late S/G2/M phases massively enter into G1/S, when they regain sensitivity to CP+dsDNA treatment. Thus, this regimen assures that no viable cells, particularly Krebs-2 TISCs, remain.
Project description:It has been established previously that up to 40% of mouse CD34(+) hematopoietic stem cells are capable of internalizing exogenous dsDNA fragments both in vivo and ex vivo. Importantly, when mice are treated with a combination of cyclophosphamide and dsDNA, the repair of interstrand crosslinks in hematopoietic progenitors is attenuated, and their pluripotency is altered. Here we show for the first time that among various actively proliferating mammalian cell populations there are subpopulations capable of internalizing dsDNA fragments. In the context of cancer, such dsDNA-internalizing cell subpopulations display cancer stem cell-like phenotype. Furthermore, using Krebs-2 ascites cells as a model, we found that upon combined treatment with cyclophosphamide and dsDNA, engrafted material loses its tumor-initiating properties which we attribute to the elimination of tumor-initiating stem cell subpopulation or loss of its tumorigenic potential.
Project description:Synchronization of biological functions to environmental signals enables organisms to anticipate and appropriately respond to daily external fluctuations and is critical to the maintenance of homeostasis. Misalignment of circadian rhythms with environmental cues is associated with adverse health outcomes. Cortisol, the downstream effector of hypothalamic-pituitary-adrenal (HPA) activity, facilitates synchronization of peripheral biological processes to the environment. Cortisol levels exhibit substantial seasonal rhythmicity, with peak levels occurring during the short-photoperiod winter months and reduced levels occurring in the long-photoperiod summer season. Seasonal changes in cortisol secretion could therefore alter its entraining capabilities, resulting in a season-dependent modification in the alignment of biological activities with the environment. We develop a mathematical model to investigate the influence of photoperiod-induced seasonal differences in the circadian rhythmicity of the HPA axis on the synchronization of the peripheral circadian clock and cell cycle in a heterogeneous cell population. Model simulations predict that the high-amplitude cortisol rhythms in winter result in the greatest entrainment of peripheral oscillators. Furthermore, simulations predict a circadian gating of the cell cycle with respect to the expression of peripheral clock genes. Seasonal differences in cortisol rhythmicity are also predicted to influence mitotic synchrony, with a high-amplitude winter rhythm resulting in the greatest synchrony and a shift in timing of the cell cycle phases, relative to summer. Our results highlight the primary interactions among the HPA axis, the peripheral circadian clock, and the cell cycle and thereby provide an improved understanding of the implications of circadian misalignment on the synchronization of peripheral regulatory processes.
Project description:BACKGROUND:Fertility in dairy cows depends on ovarian cyclicity and on uterine involution. Ovarian cyclicity and uterine involution are delayed when there is uterine dysbiosis (overgrowth of pathogenic bacteria). Fertility in dairy cows may involve a mechanism through which the uterine microbiota affects ovarian cyclicity as well as the transcriptome of the endometrium within the involuting uterus. The hypothesis was that the transcriptome of the endometrium in postpartum cows would be associated with the cyclicity status of the cow as well as the microbiota during uterine involution. The endometrium of first lactation dairy cows was sampled at 1, 5, and 9?weeks postpartum. All cows were allowed to return to cyclicity without intervention until week 5 and treated with an ovulation synchronization protocol so that sampling at week 9 was on day 13 of the estrous cycle. The endometrial microbiota was measured by 16S rRNA gene sequencing and principal component analysis. The endometrial transcriptome was measured by mRNA sequencing, differential gene expression analysis, and Ingenuity Pathway Analysis. RESULTS:The endometrial microbiota changed from week 1 to week 5 but the week 5 and week 9 microbiota were similar. The endometrial transcriptome differed for cows that were either cycling or not cycling at week 5 and cyclicity status depended in part on the endometrial microbiota. Compared with cows cycling at week 5, there were large changes in the transcriptome of cows that progressed from non-cycling at week 5 to cycling at week 9. There was evidence for concurrent and longer-term associations between the endometrial microbiota and transcriptome. The week 1 endometrial microbiota had the greatest effect on the subsequent endometrial transcriptome and this effect was greatest at week 5 and diminished by week 9. CONCLUSIONS:The cumulative response of the endometrial transcriptome to the microbiota represented the combination of past microbial exposure and current microbial exposure. The endometrial transcriptome in postpartum cows, therefore, depended on the immediate and longer-term effects of the uterine microbiota that acted directly on the uterus. There may also be an indirect mechanism through which the microbiome affects the transcriptome through the restoration of ovarian cyclicity postpartum.
Project description:Previously, we demonstrated that poorly differentiated cells of various origins, including tumor-initiating stem cells present in the ascites form of mouse cancer cell line Krebs-2, are capable of naturally internalizing both linear double-stranded DNA and circular plasmid DNA.The method of co-incubating Krebs-2 cells with extracellular plasmid DNA (pUC19) or TAMRA-5'-dUTP-labeled polymerase chain reaction (PCR) product was used. It was found that internalized plasmid DNA isolated from Krebs-2 can be transformed into competent Escherichia coli cells. Thus, the internalization processes taking place in the Krebs-2 cell subpopulation have been analyzed and compared, as assayed by E. coli colony formation assay (plasmid DNA) and cytofluorescence (TAMRA-DNA).We showed that extracellular DNA both in the form of plasmid DNA and a PCR product is internalized by the same subpopulation of Krebs-2 cells. We found that the saturation threshold for Krebs-2 ascites cells is 0.5 ?g DNA/10(6) cells. Supercoiled plasmid DNA, human high-molecular weight DNA, and 500 bp PCR fragments are internalized into the Krebs-2 tumor-initiating stem cells via distinct, non-competing internalization pathways. Under our experimental conditions, each cell may harbor 340-2600 copies of intact plasmid material, or up to 3.097 ± 0.044×10(6) plasmid copies (intact or not), as detected by quantitative PCR.The internalization dynamics of extracellular DNA, copy number of the plasmids taken up by the cells, and competition between different types of double-stranded DNA upon internalization into tumor-initiating stem cells of mouse ascites Krebs-2 have been comprehensively analyzed. Investigation of the extracellular DNA internalization into tumor-initiating stem cells is an important part of understanding their properties and possible destruction mechanisms. For example, a TAMRA-labeled DNA probe may serve as an instrument to develop a target for the therapy of cancer, aiming at elimination of tumor stem cells, as well as developing a straightforward test system for the quantification of poorly differentiated cells, including tumor-initiating stem cells, in the bulk tumor sample (biopsy or surgery specimen).
Project description:Stalagmites play an important role in paleoclimatic reconstructions from seasonal to orbital time scales as 230Th-dating can provide an accurate absolute age. Additionally, seasonal trace element and optical layers can provide a precise age. We analyzed the seasonal variability of multiple trace elements on a stalagmite (XMG) in Shihua Cave, Beijing and compared them with results from laminae counting. The results show that (1) the polished section of the topmost part of XMG has obvious bi-optical layers under a conventional transmission microscope, however, laminae are not observed using this method in the rest of the sample, and (2) The variations of P/Ca, Sr/Ca, Ba/Ca, U/Ca and Mg/Ca show seasonal cycles throughout the sample. The PC1 in the Principal Component Analysis (PCA) of five trace elements represents the annual cycle. This stalagmite was deposited over 150?±?1 years through PC1 peak counting. This result corresponds well with the annual layers and U-Th dating. Trace element cyclicity of PC1 can increase the accuracy of stalagmite dating, especially in the absence of obvious laminae and are a powerful method to identify seasonal changes in a strongly contrasting wet-dry monsoon climate region.
Project description:Felids generally follow a poly-estrous reproductive strategy. Eurasian lynx (Lynx lynx) display a different pattern of reproductive cyclicity where physiologically persistent corpora lutea (CLs) induce a mono-estrous condition which results in highly seasonal reproduction. The present study was based around a sono-morphological and endocrine study of captive Eurasian lynx, and a control-study on free-ranging lynx. We verified that CLs persist after pregnancy and pseudo-pregnancy for at least a two-year period. We could show that lynx are able to enter estrus in the following year, while CLs from the previous years persisted in structure and only temporarily reduced their function for the period of estrus onset or birth, which is unique among felids. The almost constant luteal progesterone secretion (average of 5 ng/ml serum) seems to prevent folliculogenesis outside the breeding season and has converted a poly-estrous general felid cycle into a mono-estrous cycle specific for lynx. The hormonal regulation mechanism which causes lynx to have the longest CL lifespan amongst mammals remains unclear. The described non-felid like ovarian physiology appears to be a remarkably non-plastic system. The lynx's reproductive ability to adapt to environmental and anthropogenic changes needs further investigation.
Project description:Eccentricity, obliquity, and precession are cyclic parameters of the Earth's orbit whose climatic implications have been widely demonstrated on recent and short time intervals. Amplitude modulations of these parameters on million-year time scales induce "grand orbital cycles," but the behavior and the paleoenvironmental consequences of these cycles remain debated for the Mesozoic owing to the chaotic diffusion of the solar system in the past. Here, we test for these cycles from the Jurassic to the Early Cretaceous by analyzing new stable isotope datasets reflecting fluctuations in the carbon cycle and seawater temperatures. Our results document a prominent cyclicity of ?9 My in the carbon cycle paced by changes in the seasonal dynamics of hydrological processes and long-term sea level fluctuations. These paleoenvironmental changes are linked to a great eccentricity cycle consistent with astronomical solutions. The orbital forcing signal was mainly amplified by cumulative sequestration of organic matter in the boreal wetlands under greenhouse conditions. Finally, we show that the ?9-My cycle faded during the Pliensbachian, which could either reflect major paleoenvironmental disturbances or a chaotic transition affecting this cycle.
Project description:DNA damage response (DDR) and the centrosome cycle are two of the most critical processes for maintaining a stable genome in animals. Sporadic evidence suggests a connection between these two processes. Here, we report our findings that six Fanconi Anemia (FA) proteins, including FancI and FancJ, localize to the centrosome. Intriguingly, we found that the localization of FancJ to the mother centrosome is stimulated by a DNA interstrand crosslinker, Mitomycin C (MMC). We further show that, in addition to its role in interstrand crosslinking (ICL) repair, FancJ also regulates the normal centrosome cycle as well as ICL induced centrosome amplification by activating the polo-like kinase 1 (PLK1). We have uncovered a novel function of FancJ in centrosome biogenesis and established centrosome amplification as an integral part of the ICL response.
Project description:DNA interstrand cross-links are complex lesions that covalently bind complementary strands of DNA and whose mechanism of repair remains poorly understood. In Escherichia coli, several gene products have been proposed to be involved in cross-link repair based on the hypersensitivity of mutants to cross-linking agents. However, cross-linking agents induce several forms of DNA damage, making it challenging to attribute mutant hypersensitivity specifically to interstrand cross-links. To address this, we compared the survival of UVA-irradiated repair mutants in the presence of 8-methoxypsoralen-which forms interstrand cross-links and monoadducts-to that of angelicin-a congener forming only monoadducts. We show that incision by nucleotide excision repair is not required for resistance to interstrand cross-links. In addition, neither RecN nor DNA polymerases II, IV, or V is required for interstrand cross-link survival, arguing against models that involve critical roles for double-strand break repair or translesion synthesis in the repair process. Finally, estimates based on Southern analysis of DNA fragments in alkali agarose gels indicate that lethality occurs in wild-type cells at doses producing as few as one to two interstrand cross-links per genome. These observations suggest that E. coli may lack an efficient repair mechanism for this form of damage.