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In addition to companion animals and laboratory species, about 270 carnivore species play fundamental ecological roles in different ecosystems. However, almost 40% of carnivore species are now threatened or endangered in the wild because of human activities. While protection of natural habitats is critical, it is equally important to better understand carnivore reproduction, including a solid knowledge in sperm, oocyte, and embryo biology, to maintain sustainable populations in the wild and in conservation breeding centers. Characterizing gamete and embryo biology is also needed to develop cryopreservation and assisted reproductive technologies to enhance conservation efforts. The objective of this review is to provide the most recent knowledge in the biology of sperm cells, oocytes, and early embryos across all carnivore families. Overall, most data originate from populations maintained in breeding centers or zoos. Characterizations of sperm biology and cryopreservation are far more advanced than for oocytes and embryos. Currently, sperm biology is mainly studied in Canids, Felids, Ursids, and Mustelids, with more emphasis on structural than functional properties. Importantly, fundamental studies of gamete and embryo biology in domestic dogs, cats, and ferrets have paved the way for more precise characterizations in wild counterparts as well as the development of cryopreservation and assisted reproductive technologies. A striking feature of spermatozoa across a wide range of Canids and Felids is the presence of teratospermia (60% of abnormal sperm cells), which is related to the loss of genetic diversity in some populations. Although sperm structures differ across carnivore families, sperm biology remains difficult to compare because of the small amount of data in many species. Regarding oocyte biology and embryology, data are much scarcer than in sperm cells, with too few studies going beyond structural descriptions. More carnivore species and more individuals (especially from wild populations in addition to captive ones) must be studied to improve our understanding about comparative germplasm biology and develop adequate conservation breeding strategies including the use of cryobanking and assisted reproductive technologies.

Correlation between Gli1 and PRMT1 Levels (S1); Identification of a PRMT1-Methylated Site in Gli1 (S2); Effects of R597 on Gli1 Transcriptional Activity (S3); Tumorigenicity of Wild-Type Gli1 and R597K-Mutated Gli1 (S4); Regulation of Gli1 by PRMT1 Is Independent of SMO/TGF-B/Kras (S5).

Enzymes from list of Gli1-binding proteins.

The oncogenic transcription factor Gli1 is a critical effector in the Hedgehog (Hh) pathway, which is necessary for the development and progression of pancreatic ductal adenocarcinoma (PDAC). Although TGFβ and K-Ras are known regulators of Gli1 gene transcription in this setting, it is not understood how Gli1 functional activity is regulated. Here, we report the identification of Gli1 as a substrate for the protein arginine N-methyltransferase PRMT1 in PDAC. We found that PRMT1 methylates Gli1 at R597, promoting its transcriptional activity by enhancing the binding of Gli1 to its target gene promoters. Interruption of Gli1 methylation attenuates oncogenic functions of Gli1 and sensitizes PDAC cells to gemcitabine treatment. In human PDAC specimens, the levels of both total Gli1 and methylated Gli1 were correlated positively with PRMT1 protein levels. Notably, PRMT1 regulated Gli1 independently of the canonical Hh pathway as well as the TGFβ/Kras-mediated noncanonical Hh pathway, thereby signifying a novel regulatory mechanism for Gli1 transcriptional activity. Taken together, our results identified a new posttranslational modification of Gli1 that underlies its pivotal oncogenic functions in PDAC. Cancer Res; 76(23); 7049–58. ©2016 AACR.

Gli1-binding proteins identified through mass spectrum.

Description of additional methods and procedures used in the study. Also includes Supplementary References.

PURPOSE: To evaluate the DNA integrity and developmental potential of microwave-dehydrated cat spermatozoa after storage at − 20 °C for different time periods and/or overnight shipping on dry ice. METHODS: Epididymal spermatozoa from domestic cats were microwave-dehydrated on coverslips after trehalose exposure. Dried samples were either assessed immediately, stored for various duration at − 20 °C, or shipped internationally on dry ice before continued storage. Dry-stored spermatozoa were rehydrated before assessing DNA integrity (TUNEL assays) or developmental potential (injection into in vitro matured oocytes followed by in vitro embryo culture for up to 7 days). RESULTS: Percentages of dried-rehydrated spermatozoa with intact DNA was not significantly affected (P > 0.05) by desiccation and short-term storage (range, 78.9 to 80.0%) but decreased (P 0.05). Importantly, spermatozoa shipped internationally also retained the ability to support embryo development up to the morula stage. CONCLUSION: Results demonstrated the possibility to sustain DNA integrity and developmental potential of spermatozoa by dry-preservation, even after long-term storage and long-distance shipment at non-cryogenic temperatures. While further studies are warranted, present results demonstrate that dry preservation can be a reliable approach for simple and cost-effective sperm biobanking or shipment. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10815-021-02337-4.

Long-term preservation of sperm, oocytes, and gonadal tissues at ambient temperatures has the potential to lower the costs and simplify biobanking in human reproductive medicine, as well as for the management of animal populations. Over the past decades, different dehydration protocols and long-term storage solutions at nonfreezing temperatures have been explored, mainly for mammalian sperm cells. Oocytes and gonadal tissues are more challenging to dehydrate so little to no progress have been made. Currently, the detrimental effects of the drying process itself are better characterized than the impact of long-term storage at nonfreezing temperatures. While structural and functional properties of germ cells can be preserved after dehydration, a long list of damages and stresses in nuclei, organelles, and cytoplasmic membranes have been reported and sometimes mitigated. Characterizing those damages and better understanding the response of germ cells and tissues to the stress of dehydration is fundamental. It will contribute to the development of optimal protocols while proving the safety of alternative storage options for fertility preservation. The objective of this review is to (1) document the types of damages and stress responses, as well as their mitigation in cells dried with different techniques, and (2) propose new research directions.

Anhydrous preservation is a promising approach for storage of living biomaterials at nonfreezing temperatures. Using the domestic cat model, the objectives of this study were to characterize changes in histology, DNA integrity, and viability of testicular tissues from adult versus prepubertal individuals during microwave-assisted drying. Testes from each age group were cut into small pieces before reversible membrane permeabilization, exposure to trehalose, and microwave-assisted drying during different time periods. In Experiment 1, water content was monitored for up to 40 minutes of drying. Tissues from adult or prepubertal cats experienced similar decreases of water content during the first 10 minutes. Desiccation progressed slowly between 10 and 20 minutes and then remained stable. In Experiment 2, structural properties were explored at 5, 10, and 20 minutes of desiccation. Percentages of normal seminiferous tubules were lower after 20 minutes drying in adult (43%) than in prepubertal tissues (61%). At the same time point, the proportion of cell degeneration was higher in adult (53%) than prepubertal tissues (28%). Percentages of intact DNA in tissues remained above 85% regardless of the microwave time in both age groups. Lastly, adult and prepubertal tissues only lost 33% of viability in both age groups. Collective results demonstrated for the first time that normal morphology, incidence of degeneration, DNA integrity, and viability of testicular tissues remained at acceptable levels during microwave-assisted drying for 20 minutes. Overall, prepubertal testicular tissues appeared to be more resilient to microwave-assisted desiccations than adult tissues. Importantly, water loss in the presence of trehalose after 20 minutes of desiccation already is compatible with long-term storage of testicular tissues at temperatures above -20°C, which is one step closer to future storage at supra-zero temperatures.

Rationale: Brain metastasis in patients with lung cancer is life-threatening. However, the molecular mechanism for this catastrophic disease remains elusive, and few druggable targets are available. Therefore, this study aimed to identify and characterize proteins that could be used as therapeutic targets. Methods: Proteomic analyses were conducted to identify differentially expressed membrane proteins between brain metastatic lung cancer cells and primary lung cancer cells. A neuronal growth-associated protein, brain acid soluble protein 1 (BASP1), was chosen for further investigation. The clinical relevance of BASP1 in lung adenocarcinoma was first assessed. Tyrosine kinase activity assays and in vitro and in vivo functional assays were conducted to explore the oncogenic mechanisms of BASP1. Results: The protein levels of BASP1 were positively associated with tumor progression and poor prognosis in patients with lung adenocarcinoma. Membrane-bound BASP1 increased EGFR signaling and stabilized EGFR proteins by facilitating their escape from the ubiquitin-proteasome pathway. Reciprocally, activation of EGFR recruited more BASP1 to the plasma membrane, generating a positive feedback loop between BASP1 and EGFR. Moreover, the synergistic therapeutic effects of EGFR tyrosine kinase inhibitor and arsenic trioxide led to a reduction in the level of BASP1 protein observed in lung cancer cells with acquired resistance to EGFR inhibitors. Conclusions: The reciprocal interaction between BASP1 and EGFR facilitates EGFR signaling in brain metastatic lung cancer. Targeting the newly identified BASP1-EGFR interaction could open new venues for lung cancer treatment.

Objective To present an overview of different approaches and recent advances for long-term preservation of germ cells and gonadal tissues at ambient temperatures. Methods Review of the existing literature. Results Preserving viable spermatozoa, eggs, embryos, and gonadal tissues for the long term is critical in human fertility treatment and for the management of animal populations (livestock, biomedical models, and wild species). The need and number of banked germplasms are growing very fast in all disciplines, but current storage options at freezing temperatures are often constraining and not always sustainable. Recent research indicates that structures and functions of gametes or gonadal tissues can be preserved for the long term using different strategies based on dehydration and storage at supra-zero temperatures. However, more studies are needed in rehydration and reanimation of germplasms (including proper molecular and cellular evaluations). Conclusions While a lot of research is still warranted to optimize drying and rehydration conditions for each sample type and each species, alternative preservation methods will change the paradigm in fertility preservation and biobanking. It will transform the way we maintain and manage precious biomaterials for the long term. Lay summary Living sperm cells, eggs, embryos, and reproductive tissues can be preserved at freezing temperatures for human fertility treatments and used to manage breeding in livestock, laboratory animals, and wild species through assisted reproduction. These cells can be stored in cell banks and demand for them is growing fast. However, current long-term storage options at freezing temperatures are expensive. Instead of using low temperatures, recent research indicates that these cells can be dried and stored above freezing temperatures for an extended amount of time. While a lot of research is still needed to optimize how different samples are dried and rehydrated, alternative methods of preserving cells will make fertility preservation and cell banking easier. It will also transform the way we keep and manage samples for the long term.

To participate in fertilization and embryo development, oocytes stored within the mammalian female ovary must resume meiosis as they are arrested in meiotic prophase I. This ability to resume meiosis, known as meiotic competence, requires the tight regulation of cellular metabolism and chromatin configuration. Previously, we identified nuclear proteins associated with the transition from the pre-antral to the antral follicular stage, the time at which oocytes gain meiotic competence. In this study, the objective was to specifically investigate three candidate nuclear factors: bromodomain containing protein 2 (BRD2), nucleophosmin 1 (NPM1), and asparaginase-like 1 (ASRGL1). Although these three factors have been implicated with folliculogenesis or reproductive pathologies, their requirement during oocyte maturation is unproven in any system. Experiments were conducted using different stages of oocytes isolated from adult cat ovaries. The presence of candidate factors in developing oocytes was confirmed by immunostaining. While BRD2 and ASRGL1 protein increased between pre-antral and the antral stages, changes in NPM1 protein levels between stages were not observed. Using protein inhibition experiments, we found that most BRD2 or NPM1-inhibited oocytes were incapable of participating in fertilization or embryo development. Further exploration revealed that inhibition of BRD2 and NPM-1 in cumulus-oocyte-complexes prevented oocytes from maturing to the metaphase II stage. Rather, they remained at the germinal vesicle stage or arrested shortly after meiotic resumption. We therefore have identified novel factors playing critical roles in domestic cat oocyte meiotic competence. The identification of these factors will contribute to improvement of domestic cat assisted reproduction and could serve as biomarkers of meiotically competent oocytes in other species.

The limited treatment options and therapeutic failure due to acquired resistance for patients with triple-negative breast cancer (TNBC) represent a significant challenge. Inhibitors against poly (ADP-ribose) polymerase (PARP), olaparib and talazoparib, were recently approved for the treatment of metastatic breast cancer (including TNBC) in patients with germline BRCA1/2 mutations. Despite impressive response rates of ~60%, the prolongation in median progression-free survival with a PARPi is modest, suggesting the emergence of resistance. Several studies have reported that receptor tyrosine kinases (RTKs), such as c-MET (also known as hepatocyte growth factor receptor), are involved in resistance to various anti-neoplastic agents, including PARPi. However, the mechanism by which c-MET contributes to acquired resistance to PARPi in TNBC is not fully understood. In this study, we show that hyperactivated c-Met is detected in TNBC cells with acquired resistance to PARPi, and the combination of talazoparib and crizotinib (a multi-kinase inhibitor that inhibits c-MET) synergistically inhibits proliferation in these cells. Unexpectedly, depleting c-MET had limited effect on talazoparib sensitivity in PARPi-resistant cells. Interestingly, we found evidence of epidermal growth factor receptor (EGFR) hyperactivation and interaction of EGFR/c-Met in these cells. Notably, combining EGFR and PARP inhibitors resulted in greater inhibition of proliferation in c-MET-depleted TNBC cells, and combined c-MET and EGFR inhibition increased sensitivity to talazoparib in TNBC cells with acquired resistance to PARPi. Our findings suggest that combined inhibition of c-MET and EGFR could potentially re-sensitize TNBC to the cytotoxic effects of PARPi.

Natural phenolic products from herbal medicines and dietary plants constitute the main source of lead compounds for the development of the new drug. 4,4-Dimethylcurcumin (DMCU) is a synthetic curcumin derivative and exhibits anticancer activities against breast, colon, lung, and liver cancers. However, further development of DMCU is limited by unfavorable compound properties such as very low aqueous solubility and moderate stability. To increase its solubility, we installed either or both of the ethylene-carbonate-linked L-valine side chains to DMCU phenolic groups and produced targeted 1-trifluoroacetic acid (1-TFA) and 2-trifluoroacetic acid (2-TFA) derivatives. The terminus L-valine of ethylene-carbonate-linked side chain is known to be a L-type amino acid transporter 1 (LAT1) recognition element and therefore, these two derivatives were expected to readily enter into LAT1-expressing cancer cells. In practice, 1-TFA or 2-TFA were synthesized from DMCU in four steps with 34–48% overall yield. Based on the corresponding LC-MS analysis, water solubility of DMCU, 1-TFA, and 2-TFA at room temperature (25 ± 1 °C) were 0.018, 249.7, and 375.8 mg/mL, respectively, indicating >10,000-fold higher solubility of 1-TFA and 2-TFA than DMCU. Importantly, anti-proliferative assay demonstrated that 2-TFA is a potent anti-cancer agent against LAT1-expressing lung cancer cells NCI-H460, NCI-H358, and A549 cells due to its high intracellular uptake compared to DMCU and 1-TFA. In this study, we logically designed and synthesized the targeted compounds, established the LC-MS analytical methods for evaluations of drug solubility and intracellular uptake levels, and showed improved solubility and anti-cancer activities of 2-TFA. Our results provide a strategical direction for the future development of curcuminoid-like phenolic compounds.

Understanding cellular and molecular damages in oocytes during exposure to extreme conditions is essential to optimize long-term fertility preservation approaches. Using the domestic cat (Felis catus) model, we are developing drying techniques for oocytes' germinal vesicles (GVs) as a more economical alternative to cryopreservation. The objective of the study was to characterize the influence of desiccation on nuclear envelope conformation, chromatin configuration, and the relative fluorescent intensities of histone H3 trimethylation at lysine 4 (H3K4me3) and at lysine 9 (H3K9me3) compared to vitrification. Results showed that higher proportions of dried/rehydrated GVs maintained normal nuclear envelope conformation and chromatin configuration than vitrified/warmed counterparts. Both preservation methods had a similar influence on epigenetic patterns, lowering H3K4me3 intensity to under 40% while maintaining H3K9me3 levels. Further analysis revealed that the decrease of H3K4me3 intensity mainly occurred during microwave dehydration and subsequent rehydration, whereas sample processing (permeabilization and trehalose exposure) or storage did not significantly affect the epigenetic marker. Moreover, rehydration either directly or stepwise with trehalose solutions did not influence the outcome. This is the first report demonstrating that the incidence of GV damages is lower after desiccation/rehydration than vitrification/warming.

The oncogenic transcription factor Gli1 is a critical effector in the Hedgehog (Hh) pathway, which is necessary for the development and progression of pancreatic ductal adenocarcinoma (PDAC). Although TGFβ and K-Ras are known regulators of Gli1 gene transcription in this setting, it is not understood how Gli1 functional activity is regulated. Here, we report the identification of Gli1 as a substrate for the protein arginine N-methyltransferase PRMT1 in PDAC. We found that PRMT1 methylates Gli1 at R597, promoting its transcriptional activity by enhancing the binding of Gli1 to its target gene promoters. Interruption of Gli1 methylation attenuates oncogenic functions of Gli1 and sensitizes PDAC cells to gemcitabine treatment. In human PDAC specimens, the levels of both total Gli1 and methylated Gli1 were correlated positively with PRMT1 protein levels. Notably, PRMT1 regulated Gli1 independently of the canonical Hh pathway as well as the TGFβ/Kras-mediated noncanonical Hh pathway, thereby signifying a novel regulatory mechanism for Gli1 transcriptional activity. Taken together, our results identified a new posttranslational modification of Gli1 that underlies its pivotal oncogenic functions in PDAC. Cancer Res; 76(23); 7049–58. ©2016 AACR.

Multiple mechanisms of resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been identified in EGFR-mutant non-small cell lung cancer (NSCLC); however, recurrent resistance to EGFR TKIs due to the heterogeneous mechanisms underlying resistance within a single patient remains a major challenge in the clinic. Here, we report a role of nuclear protein kinase Cδ (PKCδ) as a common axis across multiple known TKI-resistance mechanisms. Specifically, we demonstrate that TKI-inactivated EGFR dimerizes with other membrane receptors implicated in TKI resistance to promote PKCδ nuclear translocation. Moreover, the level of nuclear PKCδ is associated with TKI response in patients. The combined inhibition of PKCδ and EGFR induces marked regression of resistant NSCLC tumors with EGFR mutations.

Ovarian tissue contains large pools of immature oocytes enclosed in primordial follicles, making it an attractive target for fertility preservation in female cancer patients, livestock and wild species. Compared to cryopreservation, desiccation and long-term storage of samples at supra-zero temperatures (using strategies inspired from small organisms to resist extreme environments) would be more cost-effective and convenient. The objective of the study was to characterize the influence of microwave-assisted dehydration on structural and functional properties of living ovarian tissues. While this method allows preservation of single cells (cat oocytes and sperm cells so far) using trehalose as the xeroprotectant, it has not been developed for multicellular tissues yet. Ovarian cortex biopsies were reversibly permeabilized, exposed to various concentrations of trehalose, and dried for different times using a commercial microwave under thermal control. Effective dehydration of samples along with proper trehalose retention were reached within 30 min of microwave drying. Importantly, the process did not affect morphology and DNA integrity of follicles or stromal cells. Moreover, transcriptional activity and survival of follicles were partially maintained following 10 min of drying, which already was compatible with storage at non-cryogenic temperatures. Present data provide critical foundation to develop dry-preservation techniques for long-term storage of living multicellular tissues.

EGFR TKI-insensitive pathway confers heterogeneous mechanisms of TKI resistance via PKCδ in EGFR-mutant lung cancer. Multiple mechanisms of resistance to EGFR tyrosine kinase inhibitors (TKIs) have been identified in EGFR-mutant non-small cell lung cancer (NSCLC); however, recurrent resistance to EGFR TKIs due to the heterogeneous mechanisms underlying resistance within a single patient remains a major challenge in the clinic. Here, we report a role of EGFR heterodimer-mediated nuclear PKCδ as a common axis across multiple known TKI-resistance mechanisms. Specifically, we demonstrate that TKI-inactivated EGFR dimerizes with other membrane receptors implicated in TKI resistance to promote PKCδ nuclear translocation. Moreover, the level of nuclear PKCδ is upregulated in a significant portion of patients (~41.5%) with TKI-resistant NSCLC and is associated with TKI response in patients. The combined inhibition of PKCδ and EGFR induces marked regression of resistant NSCLC tumors with EGFR mutations. These results provide mechanistic insights into the role of the EGFR-PKCδ axis in TKI-resistance and suggest an EGFR addiction in TKI-resistant EGFR-mutant NSCLC. Citation Format: Pei-chih Lee, Mien-chie Hung. TKI insensitive role of EGFRconfers TKI resistance via PKCδ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-092.

SUMOylation of SoxE alters its recruitment of transcriptional coregulatory factors, displacing the binding of coactivators and promoting the recruitment of the corepressor Grg4., A growing number of transcriptional regulatory proteins are known to be modified by the small ubiquitin-like protein, SUMO. Posttranslational modification by SUMO may be one means by which transcriptional regulatory factors that play context-dependent roles in multiple processes can be regulated such that they direct the appropriate cellular and developmental outcomes. In early vertebrate embryos, SUMOylation of SoxE transcription factors profoundly affects their function, inhibiting their neural crest–inducing activity and promoting ear formation. In this paper, we provide mechanistic insight into how SUMO modification modulates SoxE function. We show that SUMOylation dramatically altered recruitment of transcriptional coregulator factors by SoxE proteins, displacing coactivators CREB-binding protein/p300 while promoting the recruitment of a corepressor, Grg4. These data demonstrate that SoxE proteins can function as transcriptional repressors in a SUMO-dependent manner. They further suggest a novel multivalent mechanism for SUMO-mediated recruitment of transcriptional coregulatory factors.

The efficacy of arsenic trioxide (ATO) against acute promyelocytic leukemia (APL) and relapsed APL has been well documented. ATO may cause DNA damage by generating reactive oxygen intermediates. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, modulates gene and protein expression via histone-dependent or -independent pathways that may result in chromatin decondensation, cell cycle arrest, differentiation, and apoptosis. We investigated whether ATO and SAHA act synergistically to enhance the death of cancer cells. Our current findings showed that combined treatment with ATO and SAHA resulted in enhanced suppression of non-small-cell lung carcinoma in vitro in H1299 cells and in vivo in a xenograft mouse model. Flow cytometric analysis of annexin V+ cells showed that apoptotic cell death was significantly enhanced after combined treatment with ATO and SAHA. At the doses used, ATO did not interfere with cell cycle progression, but SAHA induced p21 expression and led to G1 arrest. A Comet assay demonstrated that ATO, but not SAHA, induced DNA strand breaks in H1299 cells; however, co-treatment with SAHA significantly increased ATO-induced DNA damage. Moreover, SAHA enhanced acetylation of histone H3 and sensitized genomic DNA to DNase I digestion. Our results suggest that SAHA may cause chromatin relaxation and increase cellular susceptibility to ATO-induced DNA damage. Combined administration of SAHA and ATO may be an effective approach to the treatment of lung cancer.

A series of N-mustard-quinazoline conjugates was synthesized and subjected to antitumor studies. The N-mustard pharmacophore was attached at the C-6 of the 4-anilinoquinazolines via a urea linker. To study the structure-activity relationships of these conjugates, various substituents were introduced to the C-4 anilino moiety. The preliminary antitumor studies revealed that these agents exhibited significant antitumor activity in inhibiting various human tumor cell growths in vitro. Compounds 21b, 21g, and 21h were selected for further antitumor activity evaluation against human breast carcinoma MX-1 and prostate PC-3 xenograft in animal model. These agents showed 54-75% tumor suppression with low toxicity (5-7% body-weight changes). We also demonstrate that the newly synthesized compounds are able to induce DNA cross-linking through alkaline agarose gel shift assay and inhibited cell cycle arrest at G2/M phase.

A series of linear pyrrolo[1,2-b]isoquinoline derivatives was synthesized for antitumor evaluation. The preliminary antitumor studies reveal that both bis(hydroxymethyl) and their bis(alkylcarbamate) derivatives show significant antitumor activity in inhibiting various human tumor cell growth in vitro. 1,2-Bis(hydroxymethyl)-3-methyl-5,10-dihydropyrrolo[1,2-b]isoquinoline (20a) was selected for antitumor studies in animal models. The results show that this agent can induce complete tumor remission or significant suppression in nude mice bearing human breast (MX-1) xenograft and ovarian (SK-OV-3) xenografts, respectively. Alkaline agarose gel shifting assay showed that 20a is able to cross-link with DNA. Studies on the cell cycle inhibition revealed that this agent induces cell arrest at G2/M phase. The results warrant further antitumor investigation against other human tumor growth in animal models.

Histone deacetylase 2 (HDAC2) is a key transcriptional coregulator that is suspected to play a role during oogenesis. It is known that RNA transcription in the cat germinal vesicle (GV) stops during folliculogenesis at the late antral follicle stage and is unrelated to histone deacetylation or chromatin condensation. The objective of the present study was to determine if and how HDAC2 participates in transcription regulation in the cat GV. Spatiotemporal HDAC2 protein expression was examined by immunostaining oocytes from primary to large antral follicles. HDAC2 was detected in the majority of GVs within oocytes from early, small, and large antral follicles. At early and small antral stages, HDAC2 was found primarily in the GV's nucleoplasm. There then was a significant shift in HDAC2 localization into the nucleolus, mostly in oocytes from large antral follicles. Assessments revealed that transcription was active in oocytes that contained nucleoplasm-localized HDAC2, whereas nucleolar-bound HDAC2 was associated with loss of both global transcription and ribosomal RNA presence at all antral stages. When oocytes were exposed to the HDAC inhibitor valproic acid, results indicated that HDAC regulated transcriptional activity in the nucleoplasm, but not in the nucleolus. Collective results suggest that nucleolar translocation of HDAC2 is associated with transcriptional silencing in the GV, thereby likely contributing to an oocyte's acquisition of competence.

The ability to compact and inject the cat germinal vesicle (GV) into a recipient cytoplast allows exploration of a new fertility preservation strategy that avoids whole oocyte freezing. The objective of the present study was to understand the impact of water loss and storage time on GV DNA integrity. Immature cat oocytes were exposed to 1.5 M trehalose for 10 min before microwave-assisted dehydration for 0, 5, 10, 15, 20, 25, 30, or 40 min. Oocytes then were rehydrated to assess chromatin configuration and the incidence of DNA fragmentation (TUNEL assay). The moisture content progressively decreased (p0.05) after 40 min. Chromatin configuration was unaffected (p>0.05) over time. The percentage of GVs with DNA fragmentation was unaltered (p>0.05) from 0 to 30 min of treatment (range, 6.1%–12%), but increased (p0.05) thereafter (1 through 4 weeks, 25.0%–35.0%). Collective results demonstrate the feasibility of using microwave processing to dehydrate the mammalian GV to a moisture content that is nonlethal and enables nonfrozen storage, an alternative approach for preserving the maternal genome at cool or ambient temperature.

Histone deacetylase 2 (HDAC2) is a key transcriptional coregulator that is suspected to play a role during oogenesis. It is known that RNA transcription in the cat germinal vesicle (GV) stops during folliculogenesis at the late antral follicle stage and is unrelated to histone deacetylation or chromatin condensation. The objective of the present study was to determine if and how HDAC2 participates in transcription regulation in the cat GV. Spatiotemporal HDAC2 protein expression was examined by immunostaining oocytes from primary to large antral follicles. HDAC2 was detected in the majority of GVs within oocytes from early, small, and large antral follicles. At early and small antral stages, HDAC2 was found primarily in the GV's nucleoplasm. There then was a significant shift in HDAC2 localization into the nucleolus, mostly in oocytes from large antral follicles. Assessments revealed that transcription was active in oocytes that contained nucleoplasm-localized HDAC2, whereas nucleolar-bound HDAC2 was associated with loss of both global transcription and ribosomal RNA presence at all antral stages. When oocytes were exposed to the HDAC inhibitor valproic acid, results indicated that HDAC regulated transcriptional activity in the nucleoplasm, but not in the nucleolus. Collective results suggest that nucleolar translocation of HDAC2 is associated with transcriptional silencing in the GV, thereby likely contributing to an oocyte's acquisition of competence.

Tyrosine kinase inhibitors (TKIs) of epidermal growth factor receptor (EGFR) were the standard first-line treatments for lung cancers with activating EGFR mutations. Emergent drug resistance is the major problem in the patients who received treatment of EGFR TKIs. Second point mutation T790M mutations could be found in 50-60% patients who had drug resistance to EGFR TKIs. Alternative pathway of Met signaling, transformation to small cell lung cancer, over-expression of other HER family receptors or epithelial mesenchymal transit were reported in these patients who had drug resistance to EGFR TKIs. NFKB pathway played a role of early resistance to EGFR TKIs in the patients. The p53 mutation could be found in 70-80% lung adenocarcinoma patients. These mutations could be found in lung cancer patients who had wild type or mutated EGFR. Lung adenocarcinomas with mutated p53 lose tumor suppressor function of wild type p53 and have new function from mutated p53. We thought p53 mutation may have gain of function and result in drug resistance to EGFR TKIs in lung cancer patients. We knocked down p53 in three cancer cell line, MDA-MB-468 (p53 R273H), BT-549 (p53 R249S) and H322 (p53 R248L). We found knockdown of p53 R273H in MDA-MB-468 increased sensitivity to gefitinib. Knockdown of p53 R249S and p53 R248L showed the same response to gefitinib in cancer cell lines BT-549 and H322. Lung cancer cell line HCC827 had EGFR exon 19 deletion and was sensitive to gefitinib. We overexpressed p53 R273H, p53 R157H and p53 R249S in HCC827 lung cancer cell line. Overexpression of p53 R273H increased resistance to gefitinib in HCC827 lung cancer cell line. Overexpression of p53 R157H and p53 R249S did not increased the resistance to gefitinib. Conclusion: Gain of function of p53 R273H increased resistance to EGFR TKIs in cancer cell lines. Further immunohistochemistry staining and gene analysis of mutated p53 will be done in lung adenocarcinoma from lung cancer patients who received EGFR TKIs. Citation Format: Yueh-Fu Fang, Chun-Yu Lin, Peichih Lee. The p53 mutation R273H contributed to drug resistance of EGFR tyrosine kinase inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2940.

A series of novel 2,3-bis(hydroxymethyl)benzo[d]pyrrolo[2,1-b]thiazoles and their bis(alkylcarbamate) derivatives were synthesized starting from benzothiazole via reaction with dimethyl acetylenedicarboxylate (DMAD)/tetra-fluoro boric acid, catalytic hydrogenation, and alkylcarbamoylation. The anti-proliferative activity of these agents against human leukemia and various solid tumor cell growth in vitro was studied. The structure–activity relationship studies revealed that the bis(alkylcarbamates) derivatives are generally more cytotoxic than the corresponding bis(hydroxymethyl) congeners in inhibiting human lymphoblastic leukemia CCRF-CEM and various human solid tumor cell growth in culture. These agents have no cross-resistance to taxol or vinblastine. Studies on the therapeutic effect against human breast carcinoma MX-1 xenograft showed that complete tumor remission (CR) were achieved by treating with C1-4′-F- or C1-4′-Cl-Ph-bis(i-propylcarbamates) derivatives (19b and 19c, respectively) and more than 99% tumor suppression by the corresponding bis(ethylcarbamates) 18b and 18c at the maximal tolerated dose. Alkaline agarose gel shifting assay revealed that the newly synthesized compounds are able to induce DNA interstrand cross-linking. The present studies generated a series of new potent DNA interstrand cross-linking agents, which have potential for further antitumor drug development.

A series of novel water-soluble N-mustard-benzene conjugates bearing a urea linker were synthesized. The benzene moiety contains various hydrophilic side chains are linked to the meta- or para-position of the urea linker via a carboxamide or an ether linkage. The preliminary antitumor studies revealed that these agents exhibited potent cytotoxicity in vitro and therapeutic efficacy against human tumor xenografts in vivo. Remarkably, complete tumor remission in nude mice bearing human breast carcinoma MX-1 xenograft and significant suppression against prostate adenocarcinoma PC3 xenograft were achieved by treating with compound 9aa' at the maximum tolerable dose with relatively low toxicity. We also demonstrate that the newly synthesized compounds are able to induce DNA cross-linking through alkaline agarose gel shift assay. A pharmacokinetic profile of the representative 9aa' in rats was also investigated. The current studies suggest that this agent is a promising candidate for preclinical studies.

A series of bifunctional DNA interstrand cross-linking agents, bis(hydroxymethyl)- and bis(carbamates)-8H-3a-azacyclopenta[a]indene-1-yl derivatives were synthesized for antitumor evaluation. The preliminary antitumor studies revealed that these agents exhibited potent cytotoxicity in vitro and antitumor therapeutic efficacy against human tumor xenografts in vivo. Furthermore, these derivatives have little or no cross-resistance to either Taxol or Vinblastine. Remarkably, complete tumor remission in nude mice bearing human breast carcinoma MX-1 xenograft by 13a,b and 14g,h and significant suppression against prostate adenocarcinoma PC3 xenograft by 13b were achieved at the maximum tolerable dose with relatively low toxicity. In addition, these agents induce DNA interstrand cross-linking and substantial G2/M phase arrest in human non-small lung carcinoma H1299 cells. The current studies suggested that these agents are promising candidates for preclinical studies.

Brain asymmetry is linked with several neurological diseases, and transthyretin (TTR) is a protein sequestering β-amyloid (Aβ) and helping to prevent the Alzheimer's disease (AD). We show, by real time reverse transcription–polymerase chain reaction (RT-PCR), in situ hybridization and Western blotting, that TTR exhibits a pattern of adult male-specific, leftward distribution in the mouse brain. This asymmetry appeared to be mainly due to the asymmetric distribution of the choroid plexus cells in the ventricles. Unlike the normal mice, however, the hemispheric levels of TTR transcripts of 2- and 6-month-old Tg2576 mice, a transgenic AD mouse model overexpressing Aβ, were symmetric in both sexes. Furthermore, at the age of 10 months when the pathological AD-like features had developed, the level of TTR transcripts in the left hemisphere of the male Tg2576 became significantly lower than the right one. This lowering of TTR transcript is accompanied with a higher Aβ level in the left hemisphere of the 10-month Tg2576 males. Finally, for both genders, the TTR transcript levels in the two hemispheres of aged Tg2576 mice were lower than either the adult Tg2576 or the aged nontransgenic controls. Based on the above, we suggest scenarios to correlate the changes in the levels and hemispheric patterns of TTR expression to the pathogenesis of AD.

Many genes are recruited to the nuclear periphery upon transcriptional activation. The mechanism and functional significance of this recruitment is unclear. We find that recruitment of the yeast INO1 and GAL1 genes to the nuclear periphery is rapid and independent of transcription. Surprisingly, these genes remain at the periphery for generations after they are repressed. Localization at the nuclear periphery serves as a form of memory of recent transcriptional activation, promoting reactivation. Previously expressed GAL1 at the nuclear periphery is activated much more rapidly than long-term repressed GAL1 in the nucleoplasm, even after six generations of repression. Localization of INO1 at the nuclear periphery is necessary and sufficient to promote more rapid activation. This form of transcriptional memory is chromatin based; the histone variant H2A.Z is incorporated into nucleosomes within the recently repressed INO1 promoter and is specifically required for rapid reactivation of both INO1 and GAL1. Furthermore, H2A.Z is required to retain INO1 at the nuclear periphery after repression. Therefore, H2A.Z-mediated localization of recently repressed genes at the nuclear periphery represents an epigenetic state that confers memory of transcriptional activation and promotes reactivation., Author Summary Eukaryotic cells control the spatial arrangement of chromosomes; the localization of genes can both reflect and contribute to their transcriptional state. A number of genes in the simple eukaryote brewer's yeast are “recruited” to the nuclear periphery through interactions with the nuclear pore complex when they are expressed. The functional significance of peripheral recruitment is unclear. Here, we show that recruited genes are actively retained at the periphery for generations after transcription is repressed. This suggests that localization at the nuclear periphery represents a novel inherited state that might allow simple eukaryotic organisms to “remember” previous transcriptional activation. This type of memory allows for more robust reactivation of genes, suggesting that it is adaptive. Finally, both retention at the nuclear periphery and rapid reactivation require a variant form of histone H2A. Adaptive memory is distinct from other types of transcriptional memory. In developmental memory, transcriptional states established by transcriptional regulators early in embryogenesis are propagated long after these regulators have disappeared. Adaptive memory does not propagate a state, but represents a novel state that serves as a source of information. In this way, it resembles a rudimentary form of cellular learning that allows cells to benefit from recent experience., Recruitment of active genes to the periphery of the yeast nucleus does not require concurrent transcription.

The germinal vesicle (GV) is an alternative target to the whole oocyte for exploring new genome preservation approaches at supra-zero temperatures. Using the domestic cat model, we have demonstrated that the GV can survive simple air-drying and then be hydrated to reconstitute a viable oocyte (Graves-Herring et al., 2013). However, this method can lead to variable DNA damage due to uncontrolled water loss during the dehydration and storage processes. The objective of the present study was to better understand the role of water content during these critical steps on GV DNA integrity. Intraovarian immature oocytes collected from adult cat ovaries were denuded and permeabilized before exposure to 1.5 M trehalose for 10 min. Oocytes were transferred onto conjugate-release filters and dehydrated with a CEM SAM 255 microwave system. In Experiment 1, samples (n = 337 total oocytes, 4–5 replicates) were dehydrated at 20% power for 0, 5, 10, 15, 20, 25, 30 or 40 min. Moisture contents were assessed immediately after microwaving using volumetric Karl Fischer titration. Oocytes were rehydrated to assess chromatin configuration (Hoechst staining) and DNA fragmentation (TUNEL assay). Moisture contents progressively decreased (P 0.05) after 40 min. Chromatin configuration and proportions of degenerated oocytes (range, 2.2–9.1%) were unaffected (P > 0.05) over time. The incidence of GVs with DNA fragmentation was unaltered (P > 0.05) from 0 to 30 min of treatment (range, 6.1–12%), but increased (P 0.05) thereafter (1 through 4 weeks, 25.0–35.0%). This study characterizes, for the first time, the metrics and kinetics of moisture removal, degeneration and DNA damage related to GV desiccation as an alternative approach for preserving the maternal genome at cool or ambient temperature. Findings demonstrate the feasibility for successful microwave dehydration of the mammalian GV to reach an equilibrium moisture content that is non-lethal. Most DNA disruption occurs within the first 2 days of storage at supra-zero temperatures and more than two-thirds of GVs retain intact DNA regardless of further moisture loss during storage. Source of funding: National Institutes of Health. Conflict of interest: None declared. comizzolip@si.edu

We synthesized several novel bifunctional alkylating derivatives of 3a-aza-cyclopenta[a]indene (BO-1012, BO-1005, BO-1099, and BO-1101), which are potent DNA interstrand crosslinking agents. In in vitro cytotoxicity assay, these compounds were more cytotoxic to multidrug resistant (MDR) cells, such as KBvin10, KBtax50, and CEM/VBL, than their parental cells. Using a xenograft model, BO-1012, at a dose of 5 mg/kg, partially suppressed the growth of parental KB cells but completely suppressed the growth of KBvin10 cells in nude mice. In exploring the possible mechanism, we found that DNA double-strand break (DSB) repair activity in MDR cells, KBvin10 and CEM/VBL, was significantly reduced compared to their parental cells, KB and CEM. Reduced DSB repair activity in KBvin10 cells was likely due to a defect in nuclear translocation of DNA-PK, a component of the non-homologous end-joining repair machinery. Furthermore, BO-1012-induced DNA-PK translocation from the cytosol into the nucleus in KB cells is associated with activation of the Src/nuclear EGFR cascade, which is defective in MDR cells. Since knockdown of P-glycoprotein (P-gp) by siRNA reactivated the Src/nuclear EGFR cascade, DNA-PK translocation, and DNA repair activity in MDR cells, overexpression of P-gp attenuates the activity of DNA DSB repair via suppression of SRc/nuclear EGFR cascade. Therefore, DNA interstrand crosslinking agents may have potential therapeutic use against P-gp-overexpressing MDR cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C31.

The bioavailability of drugs is one of the important factors to determine whether a drug can be successfully developed for clinical application. Consequently, the physicochemical properties, including solubility, permeability, stability pKa, and lipophilicity/hydrophilicity balance, are key factors that influence the bioavailability of drugs. Compounds with poor solubility usually have a higher risk of difficulties to overcome during the period of new drug discovery and development because these properties may affect antitumor evaluations in animal models as well as pharmacokinetic and pharmacodynamic properties of the compound. In the present studies, we have synthesized a series of novel water-soluble N-mustard-benzene conjugates bearing a urea linker were synthesized. The benzene moiety contains a variety of hydrophilic side chains. These side chains are linked to the meta- or para-position of the urea linker via a carboxamide or an ether linkage. Compounds having a carboxamide hydrophilic side-chain can be converted into water-soluble hydrochloride or mesylate. The preliminary antitumor studies revealed that these conjugates exhibited potent cytotoxicity in vitro and antitumor therapeutic efficacy against human tumor xenografts in vivo. Furthermore, these derivatives have little or no cross-resistance to either Taxol or Vinblastine. Among these conjugates, compound WS-1707, which bearing a N,N-dimethylaminoethylcarboxamide hydrochloride water-soluble side-chain possessed potent therapeutic efficacy in nude mice bearing human breast carcinoma MX-1 (Remarkably, complete tumor remission in nude mice bearing human breast carcinoma MX-1 xenograft (complete tumor remission), prostate adenocarcinoma PC-3 xenograft (>99% suppression), and human colon cancer HCT-116 xenograft (>95% suppression) at the maximal tolerable dose 30 mg/kg, Q2D×5, intravenous injection. We also compared the therapeutic potency of WS-1707 with Carbopltin, and Doxetaxol in nude mice bearing prostate cancer HL2 xenograft (orthotopic implantation). It reveals that WS-1707 is as potent as Doxetaxol (>82% tumor suppression), but significantly more potent than Carboplatin. Study on the mechanism of action that this agent is able to induce DNA cross-linking through alkaline agarose gel shift assay. Early pharmacokinetic study shows that this conjugates has acceptable profile in rats with a half-life of 0.56 h. These results suggest that this agent is a promising candidate for preclinical studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2534. doi:10.1158/1538-7445.AM2011-2534

The efficacy of arsenic trioxide (ATO) against acute promyelocytic leukemia (APL) and relapsed APL has been well-documented. ATO may cause DNA damage through the generation of reactive oxygen intermediates. Histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), modulates gene or protein expression via histone dependent or independent pathway that may result in chromatin decondensation, cell cycle arrest, differentiation, and apoptosis. The objective of this study is to investigate whether ATO and SAHA could synergistically enhanced cell killing to cancer cells. The present studies showed that combined treatment with ATO and SAHA results in enhanced suppression of non-small cell lung carcinoma (NSCLC) H1299 cells in vitro and in xenograft mouse model. By analyzing the annexin V positive cells, apoptotic cell death is significantly enhanced in combined treatment. At the doses used, ATO does not interfere with the cell cycle progression, whereas SAHA induces the expression of p21WAF1/CIP1 and leads to G1 arrest. By the aid of Comet assay, we demonstrated that ATO induces DNA breaks in H1299 cells, but not SAHA. However, co-treatment with SAHA and ATO significantly increases ATO-induced DNA damage. Moreover, SAHA enhances acetylation of histone H3 and sensitize genomic DNA to DNase I digestion. Our present results suggest that SAHA may result in chromatin relaxation, and lead to cells become more susceptible to ATO for causing DNA damage. Therefore, combination of SAHA with ATO may provide effective approach to treatment of lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2632. doi:10.1158/1538-7445.AM2011-2632

We have synthesized several novel bifunctional alkylating derivatives of 3a-aza-cyclopenta[a]inden (BO-1012, BO-1005, BO-1099, and BO-1101) and demonstrated that they were more cytotoxic to multidrug resistant (MDR) cells, such as KBvin10, KBtax50, and CEM/VBL, than their parental cells. Using xenograft model, we confirmed that BO-1012 was more effective to suppress the growth of MDR KBvin10 cells in nude mice than to the parental KB cells. To explore the possible mechanism, we revealed that KBvin10 and CEM/VBL MDR cells have significantly lower repair activity than KB and CEM parental cells. Our results also showed that reduced repair activity in KBvin10 cells was likely due to the defective in translocation of DNA-PK, a component of repair machinery of non-homologous end-joining, from cytosol into nucleus. Furthermore, we found that BO-1012 could activate Src kinase and trigger nuclear transport of EGFR in parental cells but not in MDR cells. Inhibition of Src resulted in suppression of BO-1012-induced EGFR translocation and consequently sensitized the parental cells to BO-1012. On the contrary, Src activation induced by BO-1012 was revived in MDR cells by using siRNA to reduce the expression of P-glycoprotein (P-gp). Meanwhile, P-gp silencing also facilitated the EGFR translocation and DNA repair in MDR cells treated with BO-1012. Taken together, our results demonstrated that overexpression of P-gp in MDR cells mediates through inhibition of Src-EGFR pathway to attenuate the DNA-PK-mediated repair. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1722. doi:10.1158/1538-7445.AM2011-1722

Drug resistance is a crucial factor in the failure of cancer chemotherapy. In this study, we explored the effect of combining alkylating agents and arsenic trioxide (ATO) on the suppression of tumor cells with inherited or acquired resistance to therapeutic agents. Our results showed that combining ATO and a synthetic derivative of 3a-aza-cyclopenta[a]indenes (BO-1012), a bifunctional alkylating agent causing DNA interstrand cross-links, was more effective in killing human cancer cell lines (H460, H1299, and PC3) than combining ATO and melphalan or thiotepa. We further demonstrated that the combination treatment of H460 cells with BO-1012 and ATO resulted in severe G2/M arrest and apoptosis. In a xenograft mouse model, the combination treatment with BO-1012 and ATO synergistically reduced tumor volumes in nude mice inoculated with H460 cells. Similarly, the combination of BO-1012 and ATO effectively reduced the growth of cisplatin-resistant NTUB1/P human bladder carcinoma cells. Furthermore, the repair of BO-1012-induced DNA interstrand cross-links was significantly inhibited by ATO, and consequently, γH2AX was remarkably increased and formed nuclear foci in H460 cells treated with this drug combination. In addition, Rad51 was activated by translocating and forming foci in nuclei on treatment with BO-1012, whereas its activation was significantly suppressed by ATO. We further revealed that ATO might mediate through the suppression of AKT activity to inactivate Rad51. Taken together, the present study reveals that a combination of bifunctional alkylating agents and ATO may be a rational strategy for treating cancers with inherited or acquired drug resistance.

SummaryEpidermal growth factor receptor (EGFR) initiates a signaling cascade that leads to DNA synthesis and cell proliferation, but its role in regulating DNA replication licensing is unclear. Here, we show that activated EGFR phosphorylates the p56 isoform of Lyn, p56Lyn, at Y32, which then phosphorylates MCM7, a licensing factor critical for DNA replication, at Y600 to increase its association with other minichromosome maintenance complex proteins, thereby promoting DNA synthesis complex assembly and cell proliferation. Both p56Lyn Y32 and MCM7 Y600 phosphorylation are enhanced in proliferating cells and correlated with poor survival of breast cancer patients. These results establish a signaling cascade in which EGFR enhances MCM7 phosphorylation and DNA replication through Lyn phosphorylation in human cancer cells.