Optimization of α-amido boronic acids via cryo-electron microscopy analysis: Discovery of a novel highly selective immunoproteasome subunit LMP7 (ß5i)/LMP2 (ß1i) dual inhibitor.

The immunoproteasome subunit LMP7 (ß5i)/LMP2 (ß1i) dual blockade has been reported to suppress B cell differentiation and activation, suggesting that the dual inhibition of LMP7/LMP2 is a promising approach for treating autoimmune diseases. In contrast, the inhibition of the constitutive proteasome subunit ß5c correlates with cytotoxicity against non-immune cells. Therefore, LMP7/LMP2 dual inhibitors with high selectivity over ß5c may be desirable for treating autoimmune diseases. In this study, we present the optimization and discovery of α-amido boronic acids using cryo-electron microscopy (cryo-EM). The exploitation of structural differences between the proteasome subunits led to the identification of a highly selective LMP7/LMP2 dual inhibitor 19. Molecular dynamics simulation based on cryo-EM structures of the proteasome subunits complexed with 19 explained the inhibitory activity profile. In mice immunized with 4-hydroxy-3-nitrophenylacetyl conjugated to ovalbumin, results indicate that 19 is orally bioavailable and shows promise as potential treatment for autoimmune diseases.

Imaging Mass Spectrometry (IMS) for drug discovery and development survey: Results on methods, applications and regulatory compliance.

Imaging mass spectrometry (IMS) is increasingly used for drug discovery and development to understand target enagement, tissue distribution, drug toxicity, and disease mechanisms, etc. However, this is still a relatively new technique that requires further development validation before it will be an acceptable technique to support regulated development of new drugs. Thus, best practices will need to be established to build more confidence and gain wider acceptance by the scientific community, pharmaceutical industry, and regulatory authorities. The Imaging Mass Spectrometry Society (IMSS) and the Japan Association for Imaging Mass Spectrometry (JAIMS) have conducted a thorough survey to gather information on the current state of IMS and to identify key issues. The survey was sent to researchers or managers in the position who are currently using IMS techniques in support of their drug discovery and development efforts and/or who plan to use such tools as best practices are established. The survey probes questions related to details regarding technical aspects of IMS, which includes data acquisition, data analysis and quantitation, data integrity, reporting, applications, and regulatory concerns. This international survey was conducted online through the Survey Monkey (https://www.surveymonkey.com) in both English and Japanese from September 14 through September 30, 2020.

CERS6 required for cell migration and metastasis in lung cancer.

Sphingolipids constitute a class of bio-reactive molecules that transmit signals and exhibit a variety of physical properties in various cell types, though their functions in cancer pathogenesis have yet to be elucidated. Analyses of gene expression profiles of clinical specimens and a panel of cell lines revealed that the ceramide synthase gene CERS6 was overexpressed in non-small-cell lung cancer (NSCLC) tissues, while elevated expression was shown to be associated with poor prognosis and lymph node metastasis. NSCLC profile and in vitro luciferase analysis results suggested that CERS6 overexpression is promoted, at least in part, by reduced miR-101 expression. Under a reduced CERS6 expression condition, the ceramide profile became altered, which was determined to be associated with decreased cell migration and invasion activities in vitro. Furthermore, CERS6 knockdown suppressed RAC1-positive lamellipodia/ruffling formation and attenuated lung metastasis efficiency in mice, while forced expression of CERS6 resulted in an opposite phenotype in examined cell lines. Based on these findings, we consider that ceramide synthesis by CERS6 has important roles in lung cancer migration and metastasis.

Applications of MALDI mass spectrometry imaging for pharmacokinetic studies during drug development.

The concentration and distribution of a drug or its metabolites in tissues are key factors for understanding drug efficacy or toxicity. Conventional pharmacokinetic studies show that the plasma concentration of a drug is often unrelated to the intra-tissue concentration. Moreover, it is difficult to predict the distribution of a drug in tissues, particularly those with complex structures, even though the overall tissue concentration is measured by using homogenizing procedures. Mass spectrometry imaging (MSI) enables visualization of the spatial distribution and quantities of drugs in tissue sections without labeling, which can significantly impact on the development of new drugs and translational research. Recent advances in instrument technology and the knowledge accumulated to date could further improve the sensitivity, spatial resolution, and reproducibility of MSI. Here we present current applications of matrix-assisted laser desorption/ionization (MALDI)-MSI in pharmacokinetic imaging (PK-imaging) studies, give an overview of MALDI-MSI procedures, highlight the importance of internal standards, and give details of quantitative approaches. We also point out the need for standardizing MALDI-MSI techniques. PK-imaging using standardized MALDI-MSI methods, independent of instrument or technician expertise, is expected to contribute to acquiring reliable data in drug development and translational research in the future.

Evaluation and Comparison of Daiokanzoto and Lubiprostone for Constipation: A Retrospective Cohort Study.

Daiokanzoto (DKT) and lubiprostone (LPS) are drugs used for constipation, but few studies have compared them. This study examined the effectiveness, adverse events, and medical economic efficiency of DKT and LPS for constipation. Patients who received DKT (DKT group) and those who received LPS (LPS group) during admission to Ogaki Municipal Hospital between November 2012 and May 2016 were enrolled. Drug efficacy was evaluated based on the median value of bowel movement frequency over 1 week before and after drug administration, and their safety was evaluated by the presence or absence of diarrhea, abdominal pain, nausea, and vomiting. To assess medical economic efficiency, drug costs for constipation per week were calculated. The median values (quartile ranges) of bowel movement frequency at 1 week after drug administration were 8.5 (6.0-12.0) in the DKT group and 5 (3.0-7.0) in the LPS group, which was significantly different (p < 0.01). Diarrhea occurred significantly less often in the DKT group (4 cases) than in the LPS group (17 cases) (p < 0.01). The median cost of drugs administered for constipation for 1 week was significantly lower in the DKT group (631 [quartile range, 513-653] yen) than in the LPS group (1431 [1135-2344] yen) (p < 0.01). DKT had a higher immediate effect on constipation and was associated with more frequent bowel movement and fewer adverse events of diarrhea than LPS, suggesting that it may be effective and safe for treating constipation, and DKT is inexpensive.

Imaging mass spectrometry for toxicity assessment: a useful technique to confirm drug distribution in histologically confirmed lesions.

To evaluate the usefulness of imaging mass spectrometry (IMS) technology for assessing drug toxicity, we analyzed animal tissues in an amiodarone (AMD)-induced phospholipidosis model by IMS and confirmed the relationship between the distribution of AMD, its metabolites, and representative phospholipids (phosphatidylcholine, PC) and histological changes. AMD was administered to rats for 7 days at 150 mg/kg/day. The lung, spleen, and mesenteric lymph node were histologically examined and analyzed using IMS. The detection intensities of AMD, its metabolites, and typical PCs were higher in regions infiltrated by foamy macrophages compared with normal areas. This tendency was common in all three organs analyzed in this study. For the spleen, signals for AMD, its metabolites, and typical PCs were significantly more intense in the marginal zone, where foamy macrophages and vacuolated lymphocytes are abundant, than in the other areas. These results indicate that AMD, its metabolites, and PCs accumulate together in foamy or vacuolated cells, which is consistent with the mechanism of AMD-induced phospholipidosis. They also indicate that IMS is a useful technique for evaluating the distribution of drugs and biological components in the elucidation of toxicity mechanisms.

Efficacy and Safety of Daikenchuto for Constipation and Dose-Dependent Differences in Clinical Effects.

BACKGROUND: Daikenchuto (DKT) is a Kampo medicine used for the treatment of constipation. In this study, we evaluated the effectiveness of DKT against constipation. PATIENTS AND METHODS: Thirty-three patients administered DKT for constipation were selected and divided into low-dose (7.5 g DKT; n = 22) and high-dose (15 g DKT; n = 11) groups. We retrospectively evaluated weekly defaecation frequency, side effects, and clinical laboratory data. RESULTS: Median defaecation frequencies after DKT administration (5, 5.5, 5, and 8 for the first, second, third, and fourth weeks, resp.) were significantly higher than that before DKT administration (2) in all 33 cases (P < 0.01). One case (3%) of watery stool, one case of loose stools (3%), and no cases of abdominal pain (0%) were observed. Median defaecation frequencies in the high-dose group (7 and 9) were significantly higher than those in the low-dose group (4 and 3) in the first (P = 0.0133) and second (P = 0.0101) weeks, respectively. There was no significant change in clinical laboratory values. CONCLUSION: We suggest that DKT increases defaecation frequency and is safe for treating constipation.

Feasibility of real-time intestinal bloodstream evaluation using probe-based confocal laser endomicroscopy in a porcine intestinal ischemia model.

BACKGROUND: Intestinal ischemia can lead to fatal complications if left unrecognized during surgery. The current techniques of intraoperative microvascular assessment remain subjective. Probe-based confocal laser endomicroscopy (pCLE) has the potential to objectively evaluate microvascular blood flow in real-time setting. The present study evaluated the technical feasibility of real-time intestinal bloodstream evaluation using pCLE in a porcine intestinal ischemia model. METHODS: Seven pigs were used. The intestinal ischemia model was prepared by sequentially dividing the mesenteric blood vessels. The intestinal bloodstream was evaluated on its serosal surface using pCLE (Cellvizio 488 probe, Ultra Mini O) at every 1-cm segment from a vessel-preservation border (i.e., the cut end of the vessel). Images of the blood vessels and flow of red blood cells (RBCs) in each visualized vessel were semi-qualitatively assessed using a 3-scale scoring system. In addition, 25 surgeons blindly assessed the 10 movies recorded at 0, 1, 2, 3, and 5 cm from a vessel-preservation border using a 4-scale scoring system to confirm the consistency of the evaluation of the pCLE system. RESULTS: Images of the blood vessels were successfully obtained from the cut end of the vessel to the segment 4 cm away. Good unidirectional flow of RBCs was observed from the cut end to the 2-cm segment, whereas the flow became bidirectional between 2 and 3 cm segments. Beyond 4 cm, no flow images were obtained. The specimen obtained from the segment beyond 4 cm showed remarkable mucosal color change, which was confirmed as a necrotic change histologically. The evaluations from the cut end of the vessel to the segment 1 cm away by surgeons were excellent or good and it was almost consistent. CONCLUSIONS: Real-time bloodstream evaluation using pCLE is feasible and potentially effective for predicting intestinal ischemia during surgery.

Appropriate Follow-Up Strategies for Gastrointestinal Stromal Tumor Patients Based on the Analysis of Recurrent Interval and Patterns.

BACKGROUND/AIMS: Previous studies have proposed risk classifications for patients with gastrointestinal stromal tumor (GIST) after resection and have contributed to the prediction of its prognosis. However, optimal postoperative surveillance has not yet been established. METHODS: We retrospectively analyzed data from 115 GIST patients who experienced recurrence after complete resection. The relationships between clinicopathological characteristics and the first recurrence sites, or time to recurrence (TTR), were investigated. We also compared the characteristics between 2 subgroups based on a TTR of ≤5 or >5 years. RESULTS: The first recurrence occurred in the abdomen in 114 of 115 patients (99.1%); one case of esophageal GIST recurred in the lung. Gastric and small intestinal GISTs recurred most frequently in the liver or peritoneum, while the most common recurrences of colorectal GISTs were found to be local. Fourteen patients (12.2%) experienced recurrence after >5 years. Smaller tumors and those categorized as lower risk were significantly more frequent in the TTR >5 years group than in the TTR ≤5 years group. In the TTR >5 years group, local recurrence was the most frequent type of recurrence (42.9%). CONCLUSION: Based on abdominal examination, postoperative surveillance after complete resection for primary GISTs may be recommended for >5 years.

Efficacy of daiokanzoto in chronic constipation refractory to first-line laxatives.

There are only a few treatment options for constipation and limited evidence of suitable treatments. Daiokanzoto (DKT) is a Kampo medicine often used clincally to treat constipation. DKT is a laxative used predominantly in Japan; however, clinical data on its efficacy and safety is lacking. Patients who used DKT, but were intolerant to either magnesium oxide (MgO; MgO group; n=16) or senna extract (Senna group; n=26) were included in the present study. The frequencies of their bowel movements were compared during the 1 week prior to and following DKT administration. Within 24 hours after DKT administration, 93.8% of the patients in the MgO group evacuated their bowels. The median bowel movement frequency 1 week prior to DKT administration was 2.5 and 1 week after DKT administration was significantly increased to 7.5. In the Senna group, within 24 h of DKT administration, 80.8% of the patients evacuated their bowels. The median bowel movement frequency 1 week prior to the DKT treatment was 2.0, which significantly increased to 8.5 1 week after the administration of DKT. The adverse events from DKT treatment were mild and controllable.

Intravitreal Tissue Plasminogen Activator, Ranibizumab, and Gas Injection for Submacular Hemorrhage in Polypoidal Choroidal Vasculopathy.

PURPOSE: To investigate the efficacy of intravitreal injection of recombinant tissue plasminogen activator (rt-PA), ranibizumab, and gas without vitrectomy for submacular hemorrhage. DESIGN: Prospective, interventional, consecutive case series. PARTICIPANTS: Twenty consecutive patients (20 eyes) with submacular hemorrhage secondary to exudative age-related macular degeneration (AMD) or polypoidal choroidal vasculopathy (PCV). METHODS: Ranibizumab, rt-PA (25 µg/0.05 ml), and 100% perfluoropropane (0.3 ml) were injected intravitreally, followed by 2-day prone positioning. MAIN OUTCOME MEASURES: The primary outcome measure was best-corrected visual acuity (BCVA) 6 months after treatment. Secondary outcome measures included central retinal thickness (CRT), central pigment epithelial detachment (PED) thickness, central ellipsoid zone, recurrence rate, and complications. RESULTS: Underlying disease was exudative AMD in 1 eye and PCV in 19 eyes. Submacular hemorrhage ranged in size from 2 to 31 disc diameters. Complete displacement of submacular hemorrhage was achieved in 17 eyes (85%), and partial displacement was achieved in 3 eyes (15%). Snellen BCVA improved from 20/139 before treatment to 20/65 at 6 months (P = 0.0061). Mean change in Early Treatment Diabetic Retinopathy Study score from baseline was +13 letters (P = 0.0040). Mean CRT decreased from 599 µm before treatment to 208 µm at 6 months (P < 0.0001), and central PED thickness decreased from 188 to 88 µm (P = 0.0140). Three eyes developed vitreous hemorrhage, and 1 eye developed retinal detachment; all were treated surgically, and Snellen BCVA improved at 6 months (P = 0.0012). Recurrence was observed in 10 eyes (50%) within 6 months, but visual acuity was preserved with intravitreal injection of anti-vascular endothelial growth factor (VEGF) pro re nata (PRN). The factors that affect BCVA at 6 months after treatment were pre- and posttreatment central ellipsoid zone (P = 0.0366 and P = 0.0424), pretreatment BCVA (P = 0.0015), and pre- and posttreatment central PED thickness (P = 0.0046, P = 0.0021). CONCLUSIONS: Subretinal hemorrhage treatment by intravitreal injection of rt-PA, ranibizumab, and gas is useful to achieve hemorrhage displacement and lesion improvement. To preserve visual acuity, early detection of posttreatment recurrence and intravitreal anti-VEGF injection PRN are necessary.

Targeting ceramide synthase 6-dependent metastasis-prone phenotype in lung cancer cells.

Sphingolipids make up a family of molecules associated with an array of biological functions, including cell death and migration. Sphingolipids are often altered in cancer, though how these alterations lead to tumor formation and progression is largely unknown. Here, we analyzed non-small-cell lung cancer (NSCLC) specimens and cell lines and determined that ceramide synthase 6 (CERS6) is markedly overexpressed compared with controls. Elevated CERS6 expression was due in part to reduction of microRNA-101 (miR-101) and was associated with increased invasion and poor prognosis. CERS6 knockdown in NSCLC cells altered the ceramide profile, resulting in decreased cell migration and invasion in vitro, and decreased the frequency of RAC1-positive lamellipodia formation while CERS6 overexpression promoted it. In murine models, CERS6 knockdown in transplanted NSCLC cells attenuated lung metastasis. Furthermore, combined treatment with l-α-dimyristoylphosphatidylcholine liposome and the glucosylceramide synthase inhibitor D-PDMP induced cell death in association with ceramide accumulation and promoted cancer cell apoptosis and tumor regression in murine models. Together, these results indicate that CERS6-dependent ceramide synthesis and maintenance of ceramide in the cellular membrane are essential for lamellipodia formation and metastasis. Moreover, these results suggest that targeting this homeostasis has potential as a therapeutic strategy for CERS6-overexpressing NSCLC.

Hypoxia remodels the composition of the constituent ceramide species of HexCer and Hex2Cer with phytosphingosine and hydroxy fatty acids in human colon cancer LS174T cells.

Oxygen-requiring enzymes, such as Δ4-desaturase (dihydroceramide desaturase), sphingolipid Δ4-desaturase/C-4-hydroxylase, and fatty acid 2-hydroxylase are involved in ceramide synthesis. We prepared free ceramides, sphingomyelins and glycosphingolipids (GSLs) from cancer cells cultivated under conditions of normoxia and hypoxia, and analyzed these compounds using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Human colon cancer LS174T cells were employed because these cells highly express hydroxyl fatty acids and phytosphingosine (t18:0) which are expected to be greatly influenced by changes in oxygen levels. As expected, the populations of dihydro-species of free ceramide and sphingomyelin with C16:0 non-hydroxy fatty acid were elevated, and the populations of HexCers and Hex2Cers, composed of C16:0 or C16:0 hydroxy fatty acid (C16:0h), and sphingosine (d18:1) or t18:0, were decreased under hypoxia. However, appreciable populations of HexCer and Hex2Cer species of C24:0 or C24:0h and t18:0 remained. These results suggest that the individual species of GSLs with fatty acids possessing different alkyl chain lengths, either non-hydroxy fatty acids or hydroxyl fatty acids, may be metabolized individually.

Individual profiles of free ceramide species and the constituent ceramide species of sphingomyelin and neutral glycosphingolipid and their alteration according to the sequential changes of environmental oxygen content in human colorectal cancer Caco-2 cells.

We previously performed a systematic analysis of free ceramide (Cers) species, the constituent ceramide species of sphingomyelins and neutral glycosphingolipids (NGSLs) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with high-energy collision-induced dissociation. As a result, distinct species differences were found among Cers, sphingomyelins and NGSLs in the kidneys. Using this method, we investigated various sphingolipid species from human colon cancer Caco-2 cells as well as the influence of environmental oxygen on these species in detail. Unexpectedly, even in normoxia, all Cers species were composed of dihydrosphingosine (d18:0) and non-hydroxy fatty acid (NFA), and 34% of sphingomyelins were composed of dihydrosphingomyelins with NFA. In contrast, major constituent ceramide species of NGSLs were composed of the usual long-chain base of sphingosine (d18:1) and hydroxy fatty acid (HFA). When the cells were cultured under hypoxic condition for 3 days, all the Cers and nearly 80% of the sphingomyelins were dihydrosphingolipids composed of d18:0-NFAs, but a significant proportion of d18:1-HFAs still remained in the NGSLs. When the cells were transferred from conditions of hypoxia to normoxia again (reoxygenation), Cer species composed of d18:1-NFAs, which were not found in Cers under the original normoxic conditions, appeared. Such Cers were probably synthesized as precursors for the constituent ceramides of sphingomyelins and NGSLs.

Cells of origin of squamous epithelium, dysplasia and cancer in the head and neck region after bone marrow transplantation.

Secondary solid tumors that occur after hematopoietic stem cell transplantation (HSCT) are late complications of HSCT. Previously, secondary solid tumors were considered to be recipient-derived cells because transplanted cells do not contain epithelial cells. Recently, however, not only donor­derived epithelial cells but also donor-derived secondary solid tumors have also been reported in mice and humans. It means that circulating bone marrow-derived stem cells (BMDCs) including hematopoietic stem cells include the stem cells of many tissue types and the precancerous cells of many solid tumors. In most reports of donor-derived secondary solid tumors, however, tumors contained a low proportion of BMDC-derived epithelial cells in mixed solid tumor tissues. To our knowledge, there are only five known cases of completely donor-derived tumor tissues, i.e., four oral SCCs and a pharyngeal SCC. In this study, we analyzed five human clinical samples of solid tumors, i.e., two esophageal squamous cell carcinomas (SCCs), two oral SCCs and a tongue carcinoma. In the oral and tongue, completely donor-derived tissues were not observed, but in esophagus a completely donor-derived esophageal epidermis and SCC were observed for the first time. In addition, in another esophageal SCC patient, a completely donor-derived dysplasia region of esophageal epidermis was observed near recipient-derived SCC. This study suggests that BMDC-derived cells include the stem cells of esophageal epidermis and the precancerous cells of esophageal SCC and can differentiate into esophageal epithelium and esophageal SCC.

Self-reported assessment of health-related quality of life in children who underwent restorative proctocolectomy with ileal J-pouch anal anastomosis for ulcerative colitis.

PURPOSE: To clarify health-related quality of life (HRQOL) by self-evaluation after restorative proctocolectomy with ileal J-pouch anal anastomosis (IPAA) in children with ulcerative colitis, a questionnaire using the Pediatric Quality of Life Inventory™ 4.0 (PedsQL) was administered. METHODS: The PedsQL was administered to 13 consecutive children (mean age 14.5 years) who underwent IPAA between 2005 and 2010 in our hospital and age-matched healthy controls. The mean duration after IPAA was 2.5 years (range 0.08-6 years) at the time of this study. Healthy children completed the same questionnaire by retrospective imaging during the past 1 month by the PedsQL evaluation policy. RESULTS: Patients' total score and each functioning score after IPAA reached the same levels as those in healthy controls. Soiling, pouchitis occurrence, and bowel movements had no significant relationship to the PedsQL total score and each functioning score. CONCLUSIONS: Interference of physical activity, emotional status, and social life caused by refractory ulcerative colitis (UC) worsens patients' HRQOL. IPAA could resolve these problems in children with UC and result in an HRQOL comparable with that in healthy children.

Double balloon enteroscopy for pediatric inflammatory bowel disease.

BACKGROUND: The aim of the present study was to evaluate the efficacy and safety of double balloon enteroscopy (DBE) in children with inflammatory bowel disease (IBD). METHODS: A total of 106 DBE procedures in 67 patients were performed at Mie University Hospital from January 2008 to June 2011. Of these, 17 procedures in 12 children and adolescents with established or suspected Crohn's disease (CD) were included in the present study. The procedure, sedation, efficacy, and safety of DBE were evaluated. RESULTS: Mean patient age was 12.9 years (range, 3-19 years). Patients ≤ 15 years old had general anesthesia. The procedures included the oral approach (n= 9), the anal approach (n= 4), and the ileostomal approach (n= 4). The mean procedure duration was 60 min. Accurate diagnosis was obtained in 7/8 cases (88%) of suspected CD. Only one case was diagnosed as indeterminate colitis, although the total small and large bowel was examined on DBE and pathology. Procedure tolerance was acceptable and recovery was uneventful in all cases. No serious complications were encountered. CONCLUSIONS: With regard to the present limited IBD pediatric case series, DBE is a safe and effective procedure.

Transcriptional regulation of neutral sphingomyelinase 2 in all-trans retinoic acid-treated human breast cancer cell line, MCF-7.

Effects of all-trans retinoic acid (ATRA) on sphingomyelinase expression were examined using MCF-7 (ATRA-sensitive) and MDA-MB-231 (ATRA-resistant) breast cancer cells. Increased NSMase activity, NSMase2 mRNA and protein were observed in ATRA-treated MCF-7 but not in ATRA-treated MDA-MB-231. Increased NSMase2 mRNA of ATRA-treated MCF-7 was mostly due to enhanced transcription. Promoter analysis revealed the important 5'-promoter region of NSMase2 between -148 and -42 bp containing three Sp1 sites but no retinoic acid responsive elements. Experiments using mutated Sp1 sites of the NSMase2 promoter, Mithramycin A (a Sp inhibitor) and Sp family over-expression demonstrated the importance of Sp family protein and the three Sp1 sites for ATRA-induced NSMase2 transcription of MCF-7 cells. Although no quantitative change of bound Sp1 on NSMase2 promoter region after ATRA treatment was detected, Sp1 phosphorylation (activation) by ATRA was observed. Interestingly, PKCδ was involved in ATRA-induced increased NSMase2 transcription. ATRA-induced PKCδ phosphorylation and then activated PKCδ phosphorylated Sp1. Chromatin immunoprecipitation (ChIP) assay showed Sp1, RARα and RXRα complex formation in MCF-7 cells regardless of ATRA treatment and ATRA-induced acetylated histone H3 of the 5'-promoter. Thus, NSMase2 mRNA expression enhanced by ATRA was due to increased transcription via phosphorylated Sp1 caused by PKCδ activation, followed by chromatin remodelling with histone H3 acetylation.