Roy S. Wu

National Institutes of Health consensus development project on criteria for clinical trials in chronic graft-versus-host disease: I. Diagnosis and staging working group report.

The 2005 National Institutes of Health (NIH) Consensus Conference proposed new criteria for diagnosing and scoring the severity of chronic graft-versus-host disease (GVHD). The 2014 NIH consensus maintains the framework of the prior consensus with further refinement based on new evidence. Revisions have been made to address areas of controversy or confusion, such as the overlap chronic GVHD subcategory and the distinction between active disease and past tissue damage. Diagnostic criteria for involvement of mouth, eyes, genitalia, and lungs have been revised. Categories of chronic GVHD should be defined in ways that indicate prognosis, guide treatment, and define eligibility for clinical trials. Revisions have been made to focus attention on the causes of organ-specific abnormalities. Attribution of organ-specific abnormalities to chronic GVHD has been addressed. This paradigm shift provides greater specificity and more accurately measures the global burden of disease attributed to GVHD, and it will facilitate biomarker association studies.

Separation of basal histone synthesis from S-phase histone synthesis in dividing cells

Abstract Histone synthesis in cycling tissue-culture cells can be separated into basal synthesis and S-phase synthesis. S-phase histone synthesis comprises about 90% of total histone synthesis, and in this regard our results support the generally held model that most histone synthesis is tightly linked to DNA synthesis. Basal histone synthesis is less than 10% of the S-phase synthesis but persists throughout the S, G2 and G1 phases of the cell cycle, while S-phase synthesis rises in parallel with the increase in DNA synthesis. In Chinese hamster ovary cells, the H2A variants Z and X, and the H3 variant 3, participate in basal histone synthesis while the H2A variants 1 and 2, and the H3 variant 2, participate in S-phase histone synthesis. H2B and H4 participate in both kinds of synthesis. Because specific H2A and H3 variants are involved, basal histone synthesis cannot be attributed to contamination by cells in S-phase. Whole cells and isolated nuclei yield the same results. Basal histone synthesis can be demonstrated in cells synchronized by mitotic shake-off either alone or in combination with isoleucine starvation and colcemid blockage. It is also differentiated from S-phase histone synthesis by its relative insensitivity to hydroxyurea, indicating that basal histone synthesis is not linked or is at least less tightly linked to DNA synthesis. The basal H2A variants X and Z of five other cell lines all show similar relative insensitivities to hydroxyurea treatment. These lines include attached cultures, suspension cultures, a normal line and transformed lines from human, mouse, rat and Chinese hamster, indicating that basal histone synthesis may be a property of all cycling cells. The integration of basal histones into chromatin seems to follow the same pathway as S-phase histones, but the kinetics are somewhat slower.

Patterns of histone variant synthesis can distinguish go from G1 cells

Quiescent Chinese hamster ovary cells, as well as three other types of quiescent cells, synthesize histone at a reduced but significant rate. The variant patterns of the histone synthesis in quiescent, S-phase and G1-phase and G2-phase cells ali differed from each other. H3.3 was the only H3 variant synthesized in quiescent and in G1 and G2 cells. All four H2A variants were synthesized in quiescent and S-phase cells, but only H2A.X and H2A.Z were synthesized in G1 and G2 cells. No part of G1 or the G1-S transition could be found with an H2A synthesis pattern like that in quiescent cells. These findings suggest that the quiescent state is not part of G1, but that it is a separate and discrete state.

National Cancer Institute's First International Workshop on the Biology, Prevention, and Treatment of Relapse after Allogeneic Hematopoietic Stem Cell Transplantation: summary and recommendations from the organizing committee.

The National Cancer Institutes First International Workshop on the Biology, Prevention, and Treatment of Relapse after Allogeneic Hematopoietic Stem Cell Transplantation was organized and convened to identify, prioritize, and coordinate future research activities related to relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Each of the Workshops 6 Working Committees has published individual reports of ongoing basic, translational, and clinical research and recommended areas for future research related to the areas of relapse biology, epidemiology, prevention, and treatment. This document summarizes each committees recommendations and suggests 3 major initiatives for a coordinated research effort to address the problem of relapse after allo-HSCT: (1) to establish multicenter correlative and clinical trial networks for basic/translational, epidemiologic, and clinical research; (2) to establish a network of biorepositories for the collection of samples before and after allo-HSCT to aid in laboratory and clinical studies; and (3) to further refine, implement, and study the Workshop-proposed definitions for disease-specific response and relapse and recommendations for monitoring of minimal residual disease. These recommendations, in coordination with ongoing research initiatives and transplantation organizations, provide a research framework to rapidly and efficiently address the significant problem of relapse after allo-HSCT.

Discontinuous agarose electrophoretic system for the recovery of stained proteins from polyacrylamide gels

Abstract This paper describes two simple and efficient methods for the recovery of proteins from polyacrylamide gels stained with Coomassie brilliant blue. Both methods use ionic detergents to dissociate the stain from the protein, coupled with a discontinuous buffer system which concentrates the protein as it electrophoreses into an agarose tube gel. After electrophoresis, the piece of agarose containing the concentrated protein band is soaked in acetone which removes the salts and detergents, leaving the protein band precipitated in agarose. The sample may be used as is or may be removed from the agarose for further processing. The more general of the two methods uses SDS at alkaline pH and is useful for proteins that migrate into SDS gels. The second method uses CTAB at acid pH and is useful for histones and other proteins that migrate into acetic acid-urea-CTAB gels. A large number of different samples, as well as a large number of slices of the same spot from many polyacrylamide gels, can be processed simultaneously. A variety of proteins of different molecular weights can be purified and concentrated using this technique. Proteins recovered in this way have been used for tryptic peptide analysis.

Introduction to the Reports from the National Cancer Institute First International Workshop on the Biology, Prevention, and Treatment of Relapse after Allogeneic Hematopoietic Stem Cell Transplantation

Important advances in allogeneic hematopoietic stem cell transplantation (HSCT) over the past 20 years have substantially reduced the risk of treatment-related morbidity and mortality. However, in that same time period, the incidence of relapse has not changed significantly, despite the introduction of donor lymphocyte infusion (DLI) as a specific modality to treat relapse [1,2]. Relapse is the leading cause of death following allogeneic HSCT, and it remains the primary cause of death among patients surviving more than two year after allogeneic HSCT [1]. Moreover, the risk of relapse and disease progression is significantly higher following non-myeloablative and reduced-intensity conditioning than after myeloablative allogeneic HSCT for almost all malignant diseases for which these regimens have been employed [3–5]. Prior to the introduction of DLI, the primary approaches to the treatment of relapse were withdrawal of immune suppression, use of conventional chemotherapeutic agents, and consideration of a second allogeneic HSCT [6,7]. Initial reports demonstrated DLI was dramatically effective for relapsed chronic phase chronic myeloid leukemia (CML) and provided optimism that there was an efficacious modality which could potentially benefit the majority of patients who experienced relapse following allogeneic HSCT. This enthusiasm was quickly dampened by the subsequent reports that DLI benefited only a minority of patients with diseases other than CML [8,9]. The optimal dose, frequency, and cell type for DLI remains to be determined [9]. Various attempts have been made to augment the potency and specificity of DLI; however, the utilization and efficacy remains essentially unchanged [10–12]. The one major advancement that has occurred over the past 20 years, relative to the problem of relapse after allogeneic HSCT, is our improved understanding of the biology that underlies the graft-versus-leukemia/tumor (GVT) effect [13,14]. Research on the biology of GVT, such as the role of killer immunoglobulin receptors, could eventually have significant clinical impact [15]. Other factors, independent of GVT, also affect relapse. These include specific disease biology, tumor microenvironment, sanctuary sites and chemotherapy and/or radiotherapy resistance. How these factors interrelate to predict long-term disease control versus disease recurrence (early vs. late) is uncertain and requires further study. The experience with both syngeneic and autologous HSCT demonstrated that the conditioning regimen is important for long–term disease control [16]. However, as more and more patients are undergoing allografting with reduced-intensity and non-myeloablative conditioning regimens, disease progression and relapse will increasingly be an important cause of treatment failure. This has led to the use of strategies, such as administration of “targeted agents” and immunomodulatory agents post-transplantation to reduce the risk of relapse associated with these conditioning regimens. It was with the recognition that relapse after allogeneic HSCT is a significant clinical problem, that there is growing understanding of the mechanisms underlying the biology of relapse, and the perception there was a lack of coordination of efforts in the basic, translational and clinical research on relapse, that the idea of organizing a workshop on this subject emerged. Initially starting as an informal query, it rapidly became apparent there was significant interest in this topic and there were many individuals who were highly interested in participating in such an effort. An initial organizational meeting hosted by the National Cancer Institute (NCI) took place in San Francisco during the 2008 Annual Meeting of the American Society of Hematology. Subsequent meetings led to the formation of separate committees addressing the biology, epidemiology, prevention, monitoring and treatment of relapse. Committee chairs recruited various members both within and importantly outside the transplant community with diverse expertise relative to their specific committee topic. Each committee was given the charge of reviewing and summarizing the available scientific data on their specific topic, identifying ongoing research of great interest and potential, as well as where research was felt to be deficient. The results of these efforts were presented and discussed at a workshop sponsored by the NCI occurring November 2–3, 2009 in Bethesda, MD and included over 250 international participants. The goals of this workshop were to review the current state of the science, to present respective committee recommendations, and to have debate and discussion among all of the workshop participants with the ultimate goal of promoting a coordinated research effort to address the problem of relapse. Over the following months a summary of each committee’s findings and recommendations will be presented in the Biology of Blood and Marrow Transplantation. The purpose of these reports is multifold including providing a scientific review on various topics related to relapse after allogeneic HSCT, but more importantly to stimulate discussion and identify and prioritize research efforts on this important clinical problem.

Ubiquitinated Histones and Chromatin

Ubiquitin (Ub) is a remarkably conserved eukaryotic protein found in the cytoplasm, in the nucleus, and on the cell surface. Whereas cytoplasmic Ub is known to play an important role in proteolysis, the function of nuclear Ub is still obscure. Presumably, ubiquitination of histones affects chromatin structure, but it should be stated at the outset that we do not know how this histone modification influences transcriptional or mitotic processes. We do know, however, some aspects of the structure and metabolism of histone-Ub conjugates, and these facts should provide clues to the role of Ub in chromatin dynamics.

Fate of newly synthesized histones in G1 and G0 cells

We have shown that quiescent cells as well as those in the G1 phase of the cell cycle synthesize histones at a reduced but significant rate. Now, we show that the histones synthesized during G0 and G1 are stably incorporated into nuclei soon after synthesis. Micrococcal nuclease digestion of nuclei isolated from cells in G0 and G1 revealed that the specific histone variants synthesized in these different physiological states are found associated with DNA as nucleosomes. Nucleosomes were separated by polyacrylamide gel electrophoresis in a reducing buffer so that histone spot morphology, particularly that of the H3s was improved.

Pattern of Histone-Variant Synthesis and Implications for Gene Regulation

Studies on histone proteins impinge in two different ways on the study of transcription: (1) the role of histone proteins in the structure and function of chromatin and (2) the regulation of histone gene expression. It is generally accepted that histones serve a structural role in the packaging of the DNA in the cell nucleus (Felsenfeld, 1978). Thus, histones may affect gene expression by changing the structure of chromatin. However, the many complex and multiple posttranslational modifications of histones suggest that they have other roles in addition to purely structural ones.