Phospho-histone H3 Research Articles

Abstract 148: Neonatal Mouse Heart Regeneration is Dependent on Mononuclear Diploid Cardiomyocytes and Paracrine IGF2 Signaling

In normal mice, injury to the newborn heart is efficiently regenerated, but this capacity is lost by one week after birth when most cardiomyocytes have become post-mitotic. Concurrently during the first week, most cardiomyocytes become polyploid. Despite this temporal association, the relation of polyploidization to neonatal heart regeneration has not been adequately examined. In this study, we find that IGF2, an important mitogenic factor in heart development, is required to support neonatal mouse heart regeneration. Following injury on postnatal day 1, absence of IGF2 abolished injury-induced cell cycle entry (as indicated by phospho-histone H3 staining) during the early part of the first postnatal week, when cardiomyocytes are still mononuclear and diploid. Consequently, regeneration failed despite the later presence of additional activities that support robust cell cycle entry 7 days following injury, a time when most cardiomyocytes are polyploid and no longer able to complete cytokinesis. IGF2 originates from the endocardium/endothelium lineage and is transduced by the insulin receptor; both features distinguish the action of IGF2 in neonatal heart regeneration from that in embryonic heart development. Regeneration in injured Igf2-deficient neonates was rescued by three different contexts that elevate the percentage of mononuclear diploid cardiomyocytes beyond postnatal day 7. Thus, IGF2 is the primary paracrine-acting mitogen for heart regeneration during the early postnatal period, and IGF2-deficiency unmasks the dependence of this process on proliferation-competent mononuclear diploid cardiomyocytes.

Fractionated and Acute Proton Radiation Show Differential Intestinal Tumorigenesis and DNA Damage and Repair Pathway Response in ApcMin/+ Mice

Proton radiation is a major component of the radiation field in outer space and is used clinically in radiation therapy of resistant cancers. Although epidemiologic studies in atom bomb survivors and radiologic workers have established radiation as a risk factor for colorectal cancer (CRC), we have yet to determine the risk of CRC posed by proton radiation owing to a lack of sufficient human or animal data. The purpose of the current study was to quantitatively and qualitatively characterize differential effects of acute and fractionated high-energy protons on colorectal carcinogenesis. We used ApcMin/+ mice, a well-studied CRC model, to examine acute versus fractionated proton radiation-induced differences in intestinal tumorigenesis and associated signaling pathways. Mice were exposed to 1.88 Gy of proton radiation delivered in a single fraction or in 4 equal daily fractions (0.47 Gy × 4). Intestinal tumor number and grade were scored 100 to 110 days after irradiation, and tumor and tumor-adjacent normal tissues were harvested to assess proliferative β-catenin/Akt pathways and DNA damage response and repair pathways relevant to colorectal carcinogenesis. Significantly higher intestinal tumor number and grade, along with decreased differentiation, were observed after acute radiation relative to fractionated radiation. Acute protons induced upregulation of β-catenin and Akt pathways with increased proliferative marker phospho-histone H3. Increased DNA damage along with decreased DNA repair factors involved in mismatch repair and nonhomologous end joining were also observed after exposure to acute protons. We show increased γH2AX, 53BP1, and 8-oxo-dG, suggesting that increased ongoing DNA damage along with decreased DNA repair factors and increased proliferative responses could be triggering a higher number of intestinal tumors after acute relative to fractionated proton exposures in ApcMin/+ mice. Taken together, our data suggest greater carcinogenic potential of acute relative to fractionated proton radiation.

Abstract 2851: The effects of nidogen-1 on proliferation and migration in claudin-low mammary tumor cells

Abstract Breast cancer is the most common type of cancer among women, with one subset of the triple-negative subtype, claudin-low, known to be very aggressive and metastatic. For invasion and metastasis to occur, cancer cells must cross basement membranes, which contain structural proteins such as laminin and collagen IV and linking proteins such as perlecan and nidogen, to colonize distant tissues. Nidogen is a glycoprotein that makes up 2-3% of basement membranes and has two types: nidogen-1 (NID1) and nidogen-2 (NID2). There have been limited studies on NID1 in cancer, with results demonstrating decreased invasiveness and metastatic capabilities in Nid1 silenced cells of various cancer types. In previous work, a murine cell line representative of the claudin-low subtype, known as RJ423, was developed; it demonstrated a 5000-fold increase in Nid1 expression compared to the luminal subtypes. To test whether high Nid1 expression contributes to the aggressive, metastatic nature of claudin-low tumors, Nid1 levels were knocked down in RJ423 cells and proliferation and migratory capabilities were assessed. Phospho-histone H3 immunofluorescence demonstrated that suppressing NID1 reduced RJ423 cell proliferation significantly. Also, proliferation was explored using flow cytometry to obtain cell cycle profiles, and it was observed that there was a reduction in the G2 phase of NID1-suppressed cells. Additionally, apoptosis was assessed through flow cytometry to determine annexin V levels, but suppression of NID1 levels did not alter apoptosis. Furthermore, invasion assays demonstrated a reduction in migration through collagen IV-coated wells using NID1-suppressed cells. Knockdown of Nid1 expression in RJ423 cells reduced the level of NID1 in conditioned media, and preliminary studies suggest that the levels of NID1 in conditioned media can influence tumor cell growth. These findings suggest that NID1 could be targeted to lessen the metastatic nature of claudin-low breast cancer. Citation Format: Rebecca Jagroop, Roger Moorehead. The effects of nidogen-1 on proliferation and migration in claudin-low mammary tumor cells [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 2851.

Abstract 2554: Branched chain amino acid transaminase 1 in claudin-low breast cancer

Abstract Breast cancer, the most commonly diagnosed cancer in women, can be classified into five distinct subtypes. One subtype, claudin-low breast cancer, accounts for approximately 7% of the breast cancer cases and these tumors are aggressive and highly metastatic. RNA sequencing of human claudin-low breast cancers by other groups and RNA sequencing of a murine claudin-low mammary tumor cell line by our group has revealed that Bcat1 is significantly up-regulated in this breast cancer subtype. Bcat1 regulates the metabolism of branched chain amino acids and has been linked to numerous pathologies including heart disease, diabetes, and cancer. Based on this data we hypothesized that the expression of Bcat1 in claudin-low mammary tumors is driving the aggressive and metastatic nature of this cancer subtype and disrupting Bcat1 will deter these features. Elevated expression of Bcat1 in the murine claudin-low cell line RJ423, compared to the murine luminal mammary tumor cell line RJ345, has been confirmed at the mRNA and protein level. Bcat1 has been transiently down-regulated ~70% in RJ423 cells using siRNA and this suppression of Bcat1, contrary to the anticipated result, showed no effect on proliferation based on phospho-histone H3 immunofluorescence. However, when the cell population doubling time was investigated, a significant difference was seen between the RJ423 cell line and the cell lines with suppressed Bcat1 expression. To elucidate these contradicting results, cell cycle analysis using Bromodeoxyuridine and 7-AAD through flow cytometry is currently underway. RJ423 stably expressing Bcat1 shRNA and RJ345 stably expressing a Bcat1 expression vector are currently being selected. Further in vitro and in vivo studies will utilize these stably transfected cell lines. This study will determine whether further investigation into the effects of Bcat1 on claudin-low human breast cancer is prudent and if Bcat1 may be used as a therapeutic target. Citation Format: Lisa C. Reynen, Robert Jones, Roger Moorehead. Branched chain amino acid transaminase 1 in claudin-low breast cancer [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 2554.

Aneugen Molecular Mechanism Assay: Proof-of-Concept With 27 Reference Chemicals.

A tiered bioassay and data analysis scheme is described for elucidating the most common molecular targets responsible for chemical-induced in vitro aneugenicity: tubulin destabilization, tubulin stabilization, and inhibition of mitotic kinase(s). To evaluate this strategy, TK6 cells were first exposed to each of 27 presumed aneugens over a range of concentrations. After 4 and 24 h of treatment, γH2AX, p53, phospho-histone H3 (p-H3), and polyploidization biomarkers were evaluated using the MultiFlow DNA Damage Assay Kit. The assay identified 27 of 27 chemicals as genotoxic, with 25 exhibiting aneugenic signatures, 1 aneugenic and clastogenic, and 1 clastogenic. Subsequently, a newly described follow-up assay was employed to investigate the aneugenic agents' molecular targets. For these experiments, TK6 cells were exposed to each of 26 chemicals in the presence of 488 Taxol. After 4 h, cells were lysed and the liberated nuclei and mitotic chromosomes were stained with a nucleic acid dye and labeled with fluorescent antibodies against p-H3 and Ki-67. Flow cytometric analyses revealed that alterations to 488 Taxol-associated fluorescence were only observed with tubulin binders-increases in the case of tubulin stabilizers, decreases with destabilizers. Mitotic kinase inhibitors with known Aurora kinase B inhibiting activity were the only aneugens that dramatically decreased the ratio of p-H3-positive to Ki-67-positive nuclei. Unsupervised hierarchical clustering based on 488 Taxol fluorescence and p-H3: Ki-67 ratios clearly distinguished compounds with these disparate molecular mechanisms. Furthermore, a classification algorithm based on an artificial neural network was found to effectively predict molecular target, as leave-one-out cross-validation resulted in 25/26 agreement with a priori expectations. These results are encouraging, as they suggest that an adequate number of training set chemicals, in conjunction with a machine learning algorithm based on 488 Taxol, p-H3, and Ki-67 responses, can reliably elucidate the most commonly encountered aneugenic molecular targets.

AURKB as a target in non-small cell lung cancer with acquired resistance to anti-EGFR therapy

Non-small cell lung cancer (NSCLC) tumors harboring mutations in EGFR ultimately relapse to therapy with EGFR tyrosine kinase inhibitors (EGFR TKIs). Here, we show that resistant cells without the p.T790M or other acquired mutations are sensitive to the Aurora B (AURKB) inhibitors barasertib and S49076. Phospho-histone H3 (pH3), a major product of AURKB, is increased in most resistant cells and treatment with AURKB inhibitors reduces the levels of pH3, triggering G1/S arrest and polyploidy. Senescence is subsequently induced in cells with acquired mutations while, in their absence, polyploidy is followed by cell death. Finally, in NSCLC patients, pH3 levels are increased after progression on EGFR TKIs and high pH3 baseline correlates with shorter survival. Our results reveal that AURKB activation is associated with acquired resistance to EGFR TKIs, and that AURKB constitutes a potential target in NSCLC progressing to anti-EGFR therapy and not carrying resistance mutations.

BNP modulates cardiomyocyte proliferation in vitro and in vivo

Brain natriuretic peptide (BNP) treatment protects the heart after myocardial infarction (MI). As cardiomyocytes (CMs) express BNP receptors, the aim of this study was to determine the role of BNP on CM proliferation. We studied the BNP effect on the hearts of new born (NB) and adult infarcted mice. NB mice were injected with BNP and with BrdU two days after birth and sacrificed after eleven days. MI was induced by ligation of the left anterior descending coronary artery. BNP was injected directly into the myocardium after surgery, then intraperitoneally every 2 days. BrdU was added to drinking water. Mice were sacrificed 3 and 10 days after surgery. The infarction zone (ZI) was separated from the rest of the heart (remote zone, RZ). The number of CMs expressing BrdU + (+ 22% P = 0.04) or Aurora B+ (+ 47% P = 0.001) was increased in NB BNP-treated mice compared to the control, leading to increased number of CMs (+ 21%). Expressions of mRNAs coding for the cyclins were also increased in isolated cardiomyocytes from BNP treated hearts: cyclin E1 (+ 110% P = 0.01), A2 (+ 126% P = 0.002) and B2 (+ 70% P = 0.08). In adult infarcted mice, 3 and 10 days after MI, BNP stimulation induced an increased number of BrdU+ CMs and Aurora B + CMs in ZI (3 days: + 172% P < 0.001 and +69% P = 0.01, respectively, 10 days: + 102% P < 0.001 and + 127% P = 0.008, respectively). In parallel, CMs were isolated from NB hearts and cultured with or without BNP. After two weeks of culture, BNP treatment led to increased number of CMs (+ 21% P = 0.003), CMs expressing Aurora B (+ 58% P = 0.01), phospho-histone H3 (+ 75% P = 0.04) and Ki67 (+ 20% P = 0.03). BNP decreased CM binucleation (− 32% P = 0.001). Interestingly, BNP stimulation of CM proliferation in vitro seems to be trigger by the p38 MAPK pathway inhibition (− 42% P = 0.01). BNP treatment leads to increased number of proliferating CMs in vivo in NB hearts and in adult hearts after MI. Whether this reflects an increase of cytokinesis remains to be established in vivo.

Clinical and histopathological predictors of outcome in malignant meningioma.

We investigated possible clinical and histopathological prognostic factors in a malignant meningioma cohort with comprehensive long-term population-based follow-up data. Twenty-four consecutive patients treated surgically for malignant meningioma at the Department of Neurosurgery and the Department of Pathology, Rigshospitalet, Copenhagen, Denmark, from December 2000 to March 2014 were retrospectively evaluated regarding progression-free survival (PFS) and overall survival (OS). Clinical parameters were recorded. All specimens underwent immunohistochemical analysis for Ki-67 and phosphohistone-H3 (PHH3). Prognostication was assessed with Cox proportional hazard regression analysis. The median follow-up was 46.1months (range 0.7-150.7). The median progression-free survival was 16.5months (95% CI 11.4-43.0) and the median overall survival was 46.6months (95% CI 20.4-NA). Six patients were alive at the end of follow-up; two of these had not experienced a recurrence. No clinical parameter showed significant association with PFS or OS. Mitotic index (MI) was significantly associated with PFS and OS, and PHH3 MI with PFS. Immunohistochemical reactivity of Ki-67 > 10% was a negative predictor of PFS (HR 3.92, 95% CI 1.47-10.4, p = 0.0063) and OS (HR 3.35, 95% CI 1.12-10.1, p = 0.0313). The histological subgrouping of grade III meningioma into anaplastic and non-anaplastic revealed increased PFS for the latter (HR 4.57, CI 95% 1.32-15.7, p = 0.0164). We could not verify previous clinical parameters as prognostic factors in malignant meningioma. MI and the PHH3 MI were prognostic within WHO grade III meningiomas for PFS. An overall tumor staining of Ki-67 > 10% correlated with PFS and OS within grade III tumors.

Osthole induced apoptosis in human normal liver cells by regulating cell proliferation and endoplasmic reticulum stress.

Osthole (Ost) is often used in treatment for cancer, inflammation and rheumatism in clinic. However, Ost-induced liver injury has been reported. In this study, we aim to investigate the possible mechanism of Ost-induced hepatotoxicity in human normal liver cells (L02). When cells were exposed to Ost, the cell viability was decreased and apoptosis rate increased, the intracellular markers of oxidative stress were changed. Simultaneously, Ost altered apoptotic related proteins levels, including Bcl-2, Bax, Cleaved-Caspase-9/-8/-3, and Pro-Caspase-3/-8. In addition, Ost enhanced the levels of endoplasmic reticulum (ER) stress proteins (GRP78/Bip, CHOP, Caspase-4, IRE1α, PERK, JNK, P-JNK, and ATF4), decreased the cell proliferation and cycle-associated protein (Phospho-Histone H3, P-Cdc25C, Cdc25C, P-Cdc2, Cdc2, and Cyclin B1) level. The results show that Ost has toxic effects on L02 cells. Furthermore, it induces apoptosis by inhibiting cell proliferation, arresting cell cycle at the G2/M phase and activating ER stress.

P38\u03b1 deficiency restrains liver regeneration after partial hepatectomy triggering oxidative stress and liver injury

p38α MAPK negatively regulates the G1/S and G2/M cell cycle transitions. However, liver-specific p38α deficiency impairs cytokinesis and reduces hepatocyte proliferation during cirrhosis and aging in mice. In this work, we have studied how p38α down-regulation affects hepatocyte proliferation after partial hepatectomy, focusing on mitotic progression, cytokinesis and oxidative stress. We found that p38α deficiency triggered up-regulation of cyclins A1, B1, B2, and D1 under basal conditions and after hepatectomy. Moreover, p38α-deficient hepatocytes showed enhanced binucleation and increased levels of phospho-histone H3 but impaired phosphorylation of MNK1 after hepatectomy. The recovery of liver mass was transiently delayed in mice with p38α-deficient hepatocytes vs wild type mice. We also found that p38α deficiency caused glutathione oxidation in the liver, increased plasma aminotransferases and lactate dehydrogenase activities, and decreased plasma protein levels after hepatectomy. Interestingly, p38α silencing in isolated hepatocytes markedly decreased phospho-MNK1 levels, and silencing of either p38α or Mnk1 enhanced binucleation of hepatocytes in culture. In conclusion, p38α deficiency impairs mitotic progression in hepatocytes and restrains the recovery of liver mass after partial hepatectomy. Our results also indicate that p38α regulates cytokinesis by activating MNK1 and redox modulation.

Predictions of genotoxic potential, mode of action, molecular targets, and potency via a tiered multiflow® assay data analysis strategy.

The in vitro MultiFlow® DNA Damage Assay multiplexes γH2AX, p53, phospho-histone H3, and polyploidization biomarkers into a single flow cytometric analysis. The current report describes a tiered sequential data analysis strategy based on data generated from exposure of human TK6 cells to a previously described 85 chemical training set and a new pharmaceutical-centric test set (n = 40). In each case, exposure was continuous over a range of closely spaced concentrations, and cell aliquots were removed for analysis following 4 and 24 hr of treatment. The first data analysis step focused on chemicals' genotoxic potential, and for this purpose, we evaluated the performance of a machine learning (ML) ensemble, a rubric that considered fold increases in biomarkers against global evaluation factors (GEFs), and a hybrid strategy that considered ML and GEFs. This first tier further used ML output and/or GEFs to classify genotoxic activity as clastogenic and/or aneugenic. Test set results demonstrated the generalizability of the first tier, with particularly good performance from the ML ensemble: 35/40 (88%) concordance with a priori genotoxicity expectations and 21/24 (88%) agreement with expected mode of action (MoA). A second tier applied unsupervised hierarchical clustering to the biomarker response data, and these analyses were found to group certain chemicals, especially aneugens, according to their molecular targets. Finally, a third tier utilized benchmark dose analyses and MultiFlow biomarker responses to rank genotoxic potency. The relevance of these rankings is supported by the strong agreement found between benchmark dose values derived from MultiFlow biomarkers compared to those generated from parallel in vitro micronucleus analyses. Collectively, the results suggest that a tiered MultiFlow data analysis pipeline is capable of rapidly and effectively identifying genotoxic hazards while providing additional information that is useful for modern risk assessments-MoA, molecular targets, and potency. Environ. Mol. Mutagen. 60:513-533, 2019. © 2019 Wiley Periodicals, Inc.

The Utility of Phosphohistone H3 (PHH3) in Follicular Lymphoma Grading: A Comparative Study With Ki-67 and H&E Mitotic Count.

To determine the utility of phosphohistone H3 (PHH3) mitotic count (MC) in grading follicular lymphoma (FL). FL cases were identified (2005-2017). Three hematopathologists recorded their average Ki-67 proliferation index, MC/high-power field (hpf) using PHH3 and H&E stains, and number of centroblasts/hpf. Results were assessed for correlations and interobserver variability. Forty-three cases of FL were studied. PHH3 MC resulted in the strongest correlation to grade (r = 0.701, P < .0001) compared with Ki-67 proliferation index (PI) (r = 0.681, P < .0001) and H&E MC (r = 0.536, P = .0002) and the strongest linear relationship to centroblast count (R2 = 0.453). Agreement among pathologists was strongest for PHH3 (intraclass correlation coefficient [ICC] = 0.86) followed by Ki-67 PI (ICC = 0.85) and H&E MC (ICC = 0.78). PHH3 correlates best to histologic grade and has less interobserver variability compared with Ki-67 PI and H&E MC. These results support using PHH3 as an adjunct in FL grading.

Original article - Rho-family GTPase 1 (Rnd1) is a biomechanical stress-sensitive activator of cardiomyocyte hypertrophy

Cardiac remodeling is induced by mechanical or humoral stress causing pathological changes to the heart. Here, we aimed at identifying the role of differentially regulated genes upon dynamic mechanical stretch. Microarray of dynamic stretch induced neonatal rat ventricular cardiomyocytes (NRVCMs) discovered Rho family GTPase 1 (Rnd1) as one of the significantly upregulated genes, a cardiac role of which is not known yet. Rnd1 was consistently upregulated in NRVCMs after dynamic stretch or phenylephrine (PE) stimulation, and in a mouse model of pressure overload. Overexpression of Rnd1 in NRVCMs activated the fetal gene program (including nppa and nppb) effected into a significant increase in cell surface area in untreated, stretched or PE-treated cells. Furthermore, Rnd1 overexpression showed a positive effect on cell proliferation as detected by significant increase in Ki67, Phosphohistone H3, and EdU positive NRVCMs. Through a Yeast two-hybrid screen and immunoprecipitation analysis, we identified Myozap, an intercalated disc protein, as novel interaction partner of Rnd1. Importantly, functional analysis of this interaction revealed the importance of RND1 in the RhoA and Myozap protein network that activates serum-response factor (SRF) signaling.In summary, we identified Rnd1 as a novel stretch-sensitive gene which influences cell proliferation and cellular hypertrophy via activation of RhoA-mediated SRF dependent and independent signaling pathways.

Abstract P5-02-05: Correlation of proliferation index markers (Ki-76 and PHH3) with breast MRI apparent diffusion coefficient values

Abstract Objective: In recent literature, increasing number of studies have analyzed associations between the MRI findings and histopathological features of breast cancer. Ki-67 index is an important prognostic factor in breast cancer which provides information about the mitotic activity and the growth rate of the tumor. The proliferation index markers like Ki-67 and PHH3 (Phosphohistone H3) play an important role in decision making in the treatment regimes. PHH3 is a relatively new entity which is specific only to the proliferation giving it a unique characteristic because it is not observed at the interphase. PHH3 provides a more sensitive and accurate mitotic index with less interobserver variability when compared with conventional H&amp;E staining, thus emphasizing its potentially increased value in practice. In this study, our aim is to correlate both proliferation index markers (Ki-67 and PHH3) with breast MRI features in cases of invasive breast carcinoma. Material and Methods: In this IRB approved study, out of 126 consecutive patients' retrospective data with written consent, 29 patients (mean±std age 48.86±13.10 and range [28-82]) with full apparent diffusion coefficient (ADC), Ki67 and PHH3 data were selected. In all of these surgical specimens, immunohistochemically administered Ki-67 and PHH3 and immunopositive cells in the digital pathology were counted. MRIs were performed in a 1.5T MR unit (MagnetomEspree with Syngo MR B15 software; Siemens, Erlangen, Germany). Images were evaluated by two radiologists in consensus. Mean ADC values were obtained from radiologist drawn ROIs. The results were analyzed with principle component analysis (PCA) and pairwise covariance analysis. Results: Mean ADC value was (mean±std) 0.92±0.18 with range [0.67-1.3] (10-3mm2/s), mean Ki67 was 18.62±13.32 with range [5-62] ('%' for Ki-67) and PHH3 27.17±30.81 with a range [1-111] ('10HPF' for PHH3). When PCA was conducted in (ADC, Ki67, PHH3) space, after normalizing each of the variables 45.223% of total variance was explained mainly by the new variable obtained as a linear combination of the normalized values -0.0866*ADC - 0.9879* Ki67 - 0.7910*PHH3, 37.282% by -0.9879*ADC + 0.1545Ki67 -0.0101*PHH3 and 17.494% by 0.1283*ADC + 0.7806*Ki67 - 0.6117*PHH3 respectively. The analysis demonstrates that while Ki67 and PHH3 values explain much of the variance (45.223% + 17.494%) and thereby are highly correlated as expected, ADC on its own determines a significant portion(37.282%) of the data as well. Moreover, pairwise covariance analysis resulted in ADC, Ki67 and PHH variance values of 2.170, 1.528 and 2.197 respectively and, covariances of ADC|Ki67 (-0.1169), ADC|PHH3 (0.2048) and Ki67|PHH (1.31). Conclusion: Ki-67 and PHH3 both show good correlation in the assessment of proliferative status of breast cancer. However, breast MRI ADC values cannot be reliably used for the prediction of Ki-67 and PHH3 status of the invasive breast tumors. More studies with larger series are needed to assess the potential of breast MRI to serve as a clinical and/or prognostic factor. Citation Format: Ekemen S, Ozcan A, Aksoy Ozcan U, Ulus S, Oyan Uluc B. Correlation of proliferation index markers (Ki-76 and PHH3) with breast MRI apparent diffusion coefficient values [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-02-05.

Assessment of Effect of Intraperitoneal Tacrolimus on Liver Regeneration in Major (70%) Hepatectomy Model After Experimental Pringle Maneuver in Rats

AimSmall-for-size grafts have become more important, especially in living donor liver transplants. The Pringle maneuver, used to reduce blood loss, and the immunosuppressive medications used to prevent graft rejection in liver transplants have different side effects on liver regeneration. We researched the effect of situations where tacrolimus and the Pringle maneuver were applied or not on liver regeneration in rats with partial hepatectomy. Material and MethodsThis study was completed with 35 Wistar Albino rats. The subjects were randomly divided into 5 groups: Group 1 had the abdomen opened and no other procedure was performed; Group 2 underwent a 70% hepatectomy; Group 3 underwent a 15-minute Pringle maneuver + 70% hepatectomy; Group 4 underwent a 70% hepatectomy + 5 days of 1 mg/kg/day intraperitoneal tacrolimus; and Group 5 underwent a 150 minute Pringle maneuver + 0% hepatectomy + 5 days of 1 mg/kg/day intraperitoneal tacrolimus. All rats were sacrificed on the seventh postoperative day, remaining liver tissue was weighed, and weight indices created. The remaining liver tissue was stained with phosphohistone H3 and the mitotic index calculated. ResultsThe groups that underwent the Pringle maneuver, 70% hepatectomy, and tacrolimus administration were compared with the control group in terms of mitotic index and weight index, but no statistically significant differences were identified. ConclusionSuppression of regeneration forms a risk after liver transplantation with small-volume grafts. As a result, research on the effect of tacrolimus combined with the Pringle maneuver is important, especially for transplantations using segmented liver grafts. In our study, we showed that the use of tacrolimus had no negative effect on liver regeneration.

GSK923295 as a potential antihepatocellular carcinoma agent causing delay on liver regeneration after partial hepatectomy.

Background:The clinical trials emerged centromere protein E inhibitor GSK923295 as a promising anticancer drug, but its function in hepatocellular carcinoma (HCC) remain needs to be fully elucidated, especially as chemotherapy after hepatectomy for liver tumors. We aimed to describe anti-HCC activities of GSK923295 and compare its antiproliferative effects on liver regeneration after partial hepatectomy (PH).Methods:All subjects were randomized to treatment with either vehicle or GSK923295. Antitumor activity of GSK923295 was assessed by xenograft growth assays. The C57BL/6 mice were subjected to 70% PH and the proliferation was calculated by liver coefficient, further confirmed by immunohistochemistry. The proliferation and cell cycle analysis of liver cell AML12 and HCC cells LM3, HUH7, and HepG2 were investigated using the cell counting kit-8 assay and Flow Cytometry. The chromosome misalignment and segregation in AML12 cells were visualized by immunofluorescence.Results:Treatment with GSK923295 induced antiproliferation in HCC cell lines. It also caused delay on HCC tumor growth instead of regression both in a HCC cell line xenograft model and patient-derived tumor xenograft model. With microarray analysis, CENtromere Protein E was gradually increased in mouse liver after PH. Exposure of liver cells to GSK923295 resulted in delay on a cell cycle in mitosis with a phenotype of misaligned chromosomes and chromosomes clustered. In 70% PH mouse model, GSK923295 treatment also remarkably reduced liver regeneration in later stage, in parallel with the mitotic marker phospho-histone H3 elevation.Conclusion:The anticancer drug GSK923295 causes a significant delay on HCC tumor growth and liver regeneration after PH in later stage.

Progress of phosphohistone H3 in tumors

Phosphohistone H3 (PHH3) is one of the five newly discovered nucleocardial histones. PHH3 with other histones constitute the main protein components in chromatin in eukaryotic. For it reached the maximum value in G2 phase and M phase with mitosis, so it was considered as a specific nuclear fission marker. At present, PHH3 has been proved to be very useful to determine the nuclear fission images of tumor cells, and to distinguish the nuclear fission images from apoptotic bodies and nuclear fragments. This paper reviews the research status of PHH3 in tumors. Key words: Neoplasms; Phosphohistone H3; Mitosis

DITMD-induced mitotic defects and apoptosis in tumor cells by blocking the polo-box domain-dependent functions of polo-like kinase 1

DITMD (1, 3- Dioxolo[4,5-g] isoquinolinium 5, 6, 7, 8- tetrahydro- 4- methoxy- 6, 6- dimethyl- 5- [2- oxo- 2- (2-pyridinyl)ethyl] - iodide) is a natural product-like compound with a hydrocotarnine moiety. The aim of this study was to investigate the anticancer effects of DITMD including mitotic arrest, apoptosis, radiosensitization, and to further explore its possible mechanism. DITMD (3−30 µM) induced an obvious cell cycle delay at G2/M transition and apoptosis in HeLa cells. In a validation study, DITMD caused chromosome alignment defects and accumulation of mitotic markers such as polo-like kinase 1, cyclin B1, and phospho-histone H3. DITMD pre-treatment for 11 h also significantly decreased the cells’ survival after X-ray irradiation. In mechanism studies, DITMD inhibited the polo-box domain of polo-like kinase 1 but not the conserved kinase domain. Molecular modeling also suggests that DITMD binds at the phosphate group recognition site and inhibits the action on phospho-peptide ligands. In addition, DITMD was analyzed as a PLHSpT competitive inhibitor with an IC50 value of 2.1 μM and exhibited good selectivity against 105 distinct kinases.Taken together, these results indicate that DITMD induced chromosome alignment defects, apoptosis and radio-sensitization, and suggest that one mechanism underlying these anticancer effects involves inhibiting the polo-box domain-dependent functions of polo-like kinase 1.

Targeting EGFR of triple-negative breast cancer enhances the therapeutic efficacy of paclitaxel- and cetuximab-conjugated nanodiamond nanocomposite

Breast cancer is the most common malignancy and a leading cause of cancer-related mortality among women worldwide. Triple-negative breast cancer (TNBC) is characterized by the lack of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER2). However, epidermal growth factor receptor (EGFR) is highly expressed in most of the TNBCs, which may provide a potential target for EGFR targeting therapy. Nanodiamond (ND) is a carbon-based nanomaterial with several advantages, including fluorescence emission, biocompatibility, and drug delivery applications. In this study, we designed a nanocomposite by using ND conjugated with paclitaxel (PTX) and cetuximab (Cet) for targeting therapy on the EGFR-positive TNBC cells. ND-PTX inhibited cell viability and induced mitotic catastrophe in various human breast cancer cell lines (MDA-MB-231, MCF-7, and BT474); in contrast, ND alone did not induce cell death. ND-PTX inhibited the xenografted human breast tumors in nude mice. We further investigated ND-PTX-Cet drug efficacy on the TNBC of MDA-MB-231 breast cancer cells. ND-PTX-Cet could specifically bind to EGFR and enhanced the anticancer effects including drug uptake levels, mitotic catastrophe, and apoptosis in the EGFR-expressed MDA-MB-231 cells but not in the EGFR-negative MCF-7 cells. In addition, ND-PTX-Cet increased the protein levels of active caspase-3 and phospho-histone H3 (Ser10). Furthermore, ND-PTX-Cet showed more effective on the reduction of TNBC tumor volume by comparison with ND-PTX. Taken together, these results demonstrated that ND-PTX-Cet nanocomposite enhanced mitotic catastrophe and apoptosis by targeting EGFR of TNBC cells, which can provide a feasible strategy for TNBC therapy. Statement of significanceCurrent TNBC treatment is ineffective against the survival rate of TNBC patients. Therefore, the development of new treatment strategies for TNBC patients is urgently needed. Here, we have designed a nanocomposite by targeting on the EGFR of TNBC to enhance therapeutic efficacy by ND-conjugated PTX and Cet (ND-PTX-Cet). Interestingly, we found that the co-delivery of Cet and PTX by ND enhanced the apoptosis, mitotic catastrophe and tumor inhibition in the EGFR-expressed TNBC in vitro and in vivo. Consequently, this nanocomposite ND-PTX-Cet can be applied for targeting EGFR of human TNBC therapy.

Correction: Therapeutic effect of a novel Wnt pathway inhibitor on cardiac regeneration after myocardial infarction.

After myocardial infarction (MI), the heart is difficult to repair because of great loss of cardiomyoctyes and lack of cardiac regeneration. Novel drug candidates that aim at reducing pathological remodeling and stimulating cardiac regeneration are highly desirable. In the present study, we identified if and how a novel porcupine inhibitor CGX1321 influenced MI and cardiac regeneration. Permanent ligation of left anterior descending (LAD) coronary artery was performed in mice to induce MI injury. Cardiac function was measured by echocardiography, infarct size was examined by TTC staining. Fibrosis was evaluated with Masson's trichrome staining and vimentin staining. As a result, CGX1321 administration blocked the secretion of Wnt proteins, and inhibited both canonical and non-canonical Wnt signaling pathways. CGX1321 improved cardiac function, reduced myocardial infarct size, and fibrosis of post-MI hearts. CGX1321 significantly increased newly formed cardiomyocytes in infarct border zone of post-MI hearts, evidenced by the increased EdU+ cardiomyocytes. Meanwhile, CGX1321 increased Ki67+ and phosphohistone H3 (PH3+) cardiomyocytes in culture, indicating enhanced cardiomyocyte proliferation. The mRNA microarray showed that CGX1321 up-regulated cell cycle regulating genes such as Ccnb1 and Ccne1 CGX1321 did not alter YAP protein phosphorylation and nuclear translocation in cardiomyocytes. In conclusion, porcupine inhibitor CGX1321 reduces MI injury by limiting fibrosis and promoting regeneration. It promotes cardiomyocyte proliferation by stimulating cell cycle regulating genes with a Hippo/YAP-independent pathway.