Pharmacokinetics and pharmacodynamics of an oral formulation of decitabine and tetrahydrouridine.

BACKGROUND: Sickle cell disease (SCD) is caused by an inherited structural abnormality of adult hemoglobin causing polymerization. Fetal hemoglobin interferes with polymerization but is epigenetically silenced by DNA methyltransferase 1 (DNMT1) in adult erythropoiesis. Decitabine depletes DNMT1 and increases fetal and total hemoglobin in SCD patients, but is rapidly catabolized by cytidine deaminase (CDA) in vivo. Tetrahydrouridine (THU) inhibits CDA, safeguarding decitabine. METHODS: The pharmacokinetics and pharmacodynamics of three oral combination formulations of THU and decitabine, with different coatings producing different delays in decitabine release, were investigated in healthy participants. RESULTS: Tetrahydrouridine and decitabine were rapidly absorbed into the systemic circulation after a single combination oral dose, with relative bioavailability of decitabine ≥74% in fasted males compared with separate oral administration of THU followed by decitabine 1 h later. THU and decitabine Cmax and area under the plasma concentration versus time curve were higher in females versus males, and fasted versus fed states. Despite sex and food effect on pharmacokinetics, the pharmacodynamic effect of DNMT1 downregulation was comparable in males and females and fasted and fed states. Treatments were well tolerated. CONCLUSION: Combination oral formulations of THU with decitabine produced pharmacokinetics and pharmacodynamics suitable for oral DNMT1-targeted therapy.

Low-Dose Azacitidine with DNMT1 Level Monitoring to Treat Post-Transplantation Acute Myelogenous Leukemia or Myelodysplastic Syndrome Relapse.

Patients with early relapse of acute myelogenous leukemia (AML) or myelodysplastic syndrome (MDS) after hematopoietic cell transplantation (HCT) have a poor prognosis, and no standard treatment. Twenty-nine patients with early disease recurrence post-transplantation were treated with azacitidine (AZA; median dose, 40 mg/m2/day for 5 to 7 days). At a median follow-up of 6.3 months (range, 1.3 to 41.1 months), 7 patients (27%) had a response to AZA, defined as complete remission, hematologic improvement, or improved donor chimerism. Response occurred after a median of 3 cycles, and the median duration of response was 70 days (range, 26 to 464 days). Median survival was 6.8 months (95% confidence interval, 3.8 to 11.1 months). Survival was similar in the patients receiving an AZA dose ≤40 mg/m2 and those receiving an AZA dose >40 mg/m2. Six patients receiving donor lymphocyte infusion with AZA had a response or stable disease without worsening graft-versus-host-disease. We retrospectively used a flow cytometry assay to explore DNA-methyltransferase-1 in blood mononuclear cells as a potential pharmacodynamic marker to assess intracellular drug targeting in 8 patients. No correlation with AZA dose or response was observed. Low-dose AZA appears to have comparable efficacy to higher-dose AZA post-HCT. A significant proportion of this poor-risk population responded to low-dose AZA, suggesting a dose-independent, noncytotoxic mechanism for antileukemic activity.

UbcH7 regulates 53BP1 stability and DSB repair.

DNA double-strand break (DSB) repair is not only key to genome stability but is also an important anticancer target. Through an shRNA library-based screening, we identified ubiquitin-conjugating enzyme H7 (UbcH7, also known as Ube2L3), a ubiquitin E2 enzyme, as a critical player in DSB repair. UbcH7 regulates both the steady-state and replicative stress-induced ubiquitination and proteasome-dependent degradation of the tumor suppressor p53-binding protein 1 (53BP1). Phosphorylation of 53BP1 at the N terminus is involved in the replicative stress-induced 53BP1 degradation. Depletion of UbcH7 stabilizes 53BP1, leading to inhibition of DSB end resection. Therefore, UbcH7-depleted cells display increased nonhomologous end-joining and reduced homologous recombination for DSB repair. Accordingly, UbcH7-depleted cells are sensitive to DNA damage likely because they mainly used the error-prone nonhomologous end-joining pathway to repair DSBs. Our studies reveal a novel layer of regulation of the DSB repair choice and propose an innovative approach to enhance the effect of radiotherapy or chemotherapy through stabilizing 53BP1.

Phosphorylation of Minichromosome Maintenance 3 (MCM3) by Checkpoint Kinase 1 (Chk1) Negatively Regulates DNA Replication and Checkpoint Activation.

Mechanisms controlling DNA replication and replication checkpoint are critical for the maintenance of genome stability and the prevention or treatment of human cancers. Checkpoint kinase 1 (Chk1) is a key effector protein kinase that regulates the DNA damage response and replication checkpoint. The heterohexameric minichromosome maintenance (MCM) complex is the core component of mammalian DNA helicase and has been implicated in replication checkpoint activation. Here we report that Chk1 phosphorylates the MCM3 subunit of the MCM complex at Ser-205 under normal growth conditions. Mutating the Ser-205 of MCM3 to Ala increased the length of DNA replication track and shortened the S phase duration, indicating that Ser-205 phosphorylation negatively controls normal DNA replication. Upon replicative stress treatment, the inhibitory phosphorylation of MCM3 at Ser-205 was reduced, and this reduction was accompanied with the generation of single strand DNA, the key platform for ataxia telangiectasia mutated and Rad3-related (ATR) activation. As a result, the replication checkpoint is activated. Together, these data provide significant insights into the regulation of both normal DNA replication and replication checkpoint activation through the novel phosphorylation of MCM3 by Chk1.

A pharmacodynamic assay to monitor treatment with the hypomethylating cytosine analogs, decitabine and azacitidine.

Hypomethylating therapies using decitabine or azacitidine are actively investigated to treat acute myeloid leukemia, myelodysplastic syndromes, as maintenance therapy after allogenic stem cell transplant and hemoglobinopathies. The therapeutic mechanism is to de-repress genes that have been turned off through oncogenesis or development via methylation. The therapy can be non-cytotoxic at low dosage, sparing healthy stem cells and operating on committed precursors. Because the methods of determining maximum tolerated dose are not well suited to this paradigm, and because the mechanism of action, which is depletion of DNA methylase 1 (DNMT1), is complex and dependent on passing through a cell cycle, a pharmacodynamic assay that measures DNMT1 can inform clinical trials aimed at establishing and improving therapy. Herein, we provide an assay that measures DNMT1 relative levels in circulating T cells of peripheral blood.

Analysis of the multiparametric cell cycle data.

This chapter was originally written in 2011. The idea was to give some history of cell cycle analysis before and after flow cytometry became widely accessible; provide references to educational material for single parameter DNA content analysis, introduce and discuss multiparameter cell cycle analysis in a methodological style, and in a casual style, discuss aspects of the work over the last 40years that we have given thought, performing some experiments, but didn't publish. It feels like there is a linear progression that moves from counting cells for growth curves, to counting labeled mitotic cells by autoradiography, to DNA content analysis, to cell cycle states defined by immunofluorescence plus DNA content analysis, to extraction of cell cycle expression profiles, and finally to probability state modeling, which should be the "right" way to analyze cytometric cell cycle data. This is the sense of this chapter. In 2023, we have updated it, but the exciting, expansive aspects brought about by spectral and mass cytometry are still young and developing, and thus have not been vetted, reviewed, and presented in mature form.

Flow cytometry of DNMT1 as a biomarker of hypomethylating therapies.

The 5-azacytidine (AZA) and decitabine (DEC) are noncytotoxic, differentiation-inducing therapies approved for treatment of myelodysplastic syndrome, acute myeloid leukemias (AML), and under evaluation as maintenance therapy for AML postallogeneic hematopoietic stem cell transplant and to treat hemoglobinapathies. Malignant cell cytoreduction is thought to occur by S-phase specific depletion of the key epigenetic regulator, DNA methyltransferase 1 (DNMT1) that, in the case of cancers, thereby releases terminal-differentiation programs. DNMT1-targeting can also elevate expression of immune function genes (HLA-DR, MICA, MICB) to stimulate graft versus leukemia effects. In vivo, there is a large inter-individual variability in DEC and 5-AZA activity because of pharmacogenetic factors, and an assay to quantify the molecular pharmacodynamic effect of DNMT1-depletion is a logical step toward individualized or personalized therapy. We developed and analytically validated a flow cytometric assay for DNMT1 epitope levels in blood and bone marrow cell subpopulations defined by immunophenotype and cell cycle state. Wild type (WT) and DNMT1 knock out (DKO) HC116 cells were used to select and optimize a highly specific DNMT1 monoclonal antibody. Methodologic validation of the assay consisted of cytometry and matching immunoblots of HC116-WT and -DKO cells and peripheral blood mononuclear cells; flow cytometry of H116-WT treated with DEC, and patient samples before and after treatment with 5-AZA. Analysis of patient samples demonstrated assay reproducibility, variation in patient DNMT1 levels prior to treatment, and DNMT1 depletion posttherapy. A flow-cytometry assay has been developed that in the research setting of clinical trials can inform studies of DEC or 5-AZA treatment to achieve targeted molecular pharmacodynamic effects and better understand treatment-resistance/failure.

High-resolution kinetics of cytokine signaling in human CD34/CD117-positive cells in unfractionated bone marrow.

Cytokine-mediated phosphorylation of Erk (pErk), ribosomal S6 (pS6), and Stat5 (pStat5) in CD34(+)/CD117(+) blast cells in normal bone marrow from 9 healthy adult donors were analyzed over 60 minutes. Treatment with stem cell factor (SCF), Flt3-ligand (FL), IL-3, and GM-CSF and measurement by multiparametric flow cytometry yielded distinctive, highly uniform phosphoprotein kinetic profiles despite a diverse sample population. The correlated responses for SCF- and FL-stimulated pErk and pS6 were similar. Half the population phosphorylated Erk in response to SCF between 0.9 and 1.2 minutes, and S6 phosphorylation followed approximately a minute later (t½(pS6 rise) = 2.2-2.7 minutes). The FL response was equally fast but more variable (t½(pErk rise) = 0.9-1.3 minutes; t½(pS6 rise) = 2.5-3.5 minutes). Stat5 was not activated in 97% of the cells by either cytokine. IL-3 and GM-CSF were similar to each other with half of blast cells phosphorylating Stat5 and 15% to 20% responding through Erk and S6. Limited comparison with leukemic blasts confirmed universal abnormal signaling in AML that is significantly different from normal bone marrow blasts. These differences included sustained signals, a larger fraction of responding cells, and amplification of phosphorylation levels for at least one phosphoprotein. These data support the eventual use of this approach for disease diagnosis and monitoring.

Duffy antigen is expressed during erythropoiesis in Duffy-negative individuals.

The erythrocyte silent Duffy blood group phenotype in Africans is thought to confer resistance to Plasmodium vivax blood-stage infection. However, recent studies report P. vivax infections across Africa in Fy-negative individuals. This suggests that the globin transcription factor 1 (GATA-1) SNP underlying Fy negativity does not entirely abolish Fy expression or that P. vivax has developed a Fy-independent red blood cell (RBC) invasion pathway. We show that RBCs and erythroid progenitors from in vitro differentiated CD34 cells and from bone marrow aspirates from Fy-negative samples express a functional Fy on their surface. This suggests that the GATA-1 SNP does not entirely abolish Fy expression. Given these results, we developed an in vitro culture system for P. vivax and show P. vivax can invade erythrocytes from Duffy-negative individuals. This study provides evidence that Fy is expressed in Fy-negative individuals and explains their susceptibility to P. vivax with major implications and challenges for P. vivax malaria eradication.

A hybrid model of mammalian cell cycle regulation.

The timing of DNA synthesis, mitosis and cell division is regulated by a complex network of biochemical reactions that control the activities of a family of cyclin-dependent kinases. The temporal dynamics of this reaction network is typically modeled by nonlinear differential equations describing the rates of the component reactions. This approach provides exquisite details about molecular regulatory processes but is hampered by the need to estimate realistic values for the many kinetic constants that determine the reaction rates. It is difficult to estimate these kinetic constants from available experimental data. To avoid this problem, modelers often resort to 'qualitative' modeling strategies, such as Boolean switching networks, but these models describe only the coarsest features of cell cycle regulation. In this paper we describe a hybrid approach that combines the best features of continuous differential equations and discrete Boolean networks. Cyclin abundances are tracked by piecewise linear differential equations for cyclin synthesis and degradation. Cyclin synthesis is regulated by transcription factors whose activities are represented by discrete variables (0 or 1) and likewise for the activities of the ubiquitin-ligating enzyme complexes that govern cyclin degradation. The discrete variables change according to a predetermined sequence, with the times between transitions determined in part by cyclin accumulation and degradation and as well by exponentially distributed random variables. The model is evaluated in terms of flow cytometry measurements of cyclin proteins in asynchronous populations of human cell lines. The few kinetic constants in the model are easily estimated from the experimental data. Using this hybrid approach, modelers can quickly create quantitatively accurate, computational models of protein regulatory networks in cells.

Major differences in the responses of primary human leukocyte subsets to IFN-beta.

Treatment of cell lines with type I IFNs activates the formation of IFN-stimulated gene factor 3 (STAT1/STAT2/IFN regulatory factor-9), which induces the expression of many genes. To study this response in primary cells, we treated fresh human blood with IFN-ß and used flow cytometry to analyze phosphorylated STAT1, STAT3, and STAT5 in CD4(+) and CD8(+) T cells, B cells, and monocytes. The activation of STAT1 was remarkably different among these leukocyte subsets. In contrast to monocytes and CD4(+) and CD8(+) T cells, few B cells activated STAT1 in response to IFN-ß, a finding that could not be explained by decreased levels of IFNAR2 or STAT1 or enhanced levels of suppressor of cytokine signaling 1 or relevant protein tyrosine phosphatases in B cells. Microarray and real-time PCR analyses revealed the induction of STAT1-dependent proapoptotic mRNAs in monocytes but not in B cells. These data show that IFN-stimulated gene factor 3 or STAT1 homodimers are not the main activators of gene expression in primary B cells of healthy humans. Notably, in B cells and, especially in CD4(+) T cells, IFN-ß activated STAT5 in addition to STAT3, with biological effects often opposite from those driven by activated STAT1. These data help to explain why IFN-ß increases the survival of primary human B cells and CD4(+) T cells but enhances the apoptosis of monocytes, as well as to understand how leukocyte subsets are differentially affected by endogenous type I IFNs during viral or bacterial infections and by type I IFN treatment of patients with multiple sclerosis, hepatitis, or cancer.

DNA synthesis from unbalanced nucleotide pools causes limited DNA damage that triggers ATR-CHK1-dependent p53 activation.

p53-dependent G(1) and G(2) cell cycle checkpoints are activated in response DNA damage that help to maintain genomic stability. p53 also helps to protect cells from damage that occurs during S phase, for example, when the cells are starved for DNA precursors or irradiated with a low dose of UV. p53 is activated in normal cells starved for pyrimidine nucleotides by treatment with N-(phosphonacetyl)-l-aspartate (PALA). The treated cells progress through a first S phase with kinetics similar to those of untreated cells. However, the DNA of the treated cells begins to become damaged rapidly, within 12 h, as revealed by a comet assay, which detects broken DNA, and by staining for phosphorylated histone H2AX, which accumulates at sites of DNA damage. Because the cells survive, the damage must be reversible, suggesting single-strand breaks or gaps as the most likely possibility. The transiently damaged DNA stimulates activation of ATR and CHK1, which in turn catalyze the phosphorylation and accumulation of p53. Although PALA-induced DNA damage occurs only in dividing cells, the p53 that is activated is only competent to transcribe genes such as p21 and macrophage inhibitory cytokine 1 (whose products regulate G(2) and G(1) or S phase checkpoints, respectively) after the cells have exited the S phase during which damage occurs. We propose that p53 is activated by stimulation of mismatch repair in response to the misincorporation of deoxynucleotides into newly synthesized DNA, long before the lack of pyrimidine nucleoside triphosphates causes the rate of DNA synthesis to slow appreciably.

Cytometry of chromatin bound Mcm6 and PCNA identifies two states in G1 that are separated functionally by the G1 restriction point.

BACKGROUND: Cytometric measurements of DNA content and chromatin-bound Mcm2 have demonstrated bimodal patterns of expression in G1. These patterns, the replication licensing function of Mcm proteins, and a correlation between Mcm loading and cell cycle commitment for cells re-entering the cell cycle, led us to test the idea that cells expressing a defined high level of chromatin-bound Mcm6 in G1 are committed--i.e., past the G1 restriction point. We developed a cell-based assay for tightly-bound PCNA (PCNA*) and Mcm6 (Mcm6*), DNA content, and a mitotic marker to clearly define G1, S, G2, and M phases of the cell cycle. hTERT-BJ1, hTERT-RPE-1, and Molt4 cells were extracted with Triton X-100 followed by methanol fixation, stained with antibodies and DAPI, then measured by cytometry. RESULTS: Bivariate analysis of cytometric data demonstrated complex patterns with distinct clustering for all combinations of the 4 variables. In G1, cells clustered in two groups characterized by low and high Mcm6* expression. Serum starvation and release experiments showed that residence in the high group was in late G1, just prior to S phase. Kinetic experiments, employing serum withdrawal, and stathmokinetic analysis with aphidicolin, mimosine or nocodazole demonstrated that cells with high levels of Mcm6* cycled with the committed phases of the cell cycle (S, G2, and M). CONCLUSIONS: A multivariate assay for Mcm6*, PCNA*, DNA content, and a mitotic marker provides analysis capable of estimating the fraction of pre and post-restriction point G1 cells and supports the idea that there are at least two states in G1 defined by levels of chromatin bound Mcm proteins.

Phase II study of bryostatin 1 and vincristine for aggressive non-Hodgkin lymphoma relapsing after an autologous stem cell transplant.

Bryostatin 1, isolated from a marine bryozoan, enhances the efficacy of cytotoxic agents through modulation of the protein kinase C pathway and is active in combination with vincristine for diffuse large B-cell lymphoma. Further, the apoptotic frequency of peripheral blood T lymphocytes as determined by flow cytometry may predict which patients will respond to this combination. We tested the efficacy and safety of bryostatin 1 50 microg/m(2) given over 24 hr and vincristine 1.4 mg/m(2) on days 1 and 15 every 28 days in aggressive B-cell non-Hodgkin lymphoma (NHL) relapsing after autologous stem cell transplantation. End points included tumor response, toxicity, and survival. Responses were correlated with an increase in apoptotic frequency of CD5+ cells by flow cytometry using annexin V staining. Fourteen patients were enrolled with 13 being evaluable for a response. The overall response rate was 31% with two patients achieving a complete response. The most common toxicities were Grade 3 lymphopenia (seven patients), Grade 3 to 4 neutropenia (two patients), and Grade 3 hypophosphatemia (two patients). Median progression-free and overall survivals for all patients were 5.7 and 21.4 months, respectively. One patient demonstrated an increase in T-cell apoptotic frequency, also achieving a complete response. Bryostatin 1 and vincristine have efficacy in select patients with aggressive NHL. Future investigations of agents targeting the protein kinase C pathway may benefit from early response assessment using flow cytometry to evaluate T-cell apoptosis.

Monitoring Plasmodium falciparum growth and development by UV flow cytometry using an optimized Hoechst-thiazole orange staining strategy.

The complex life cycle of Plasmodium falciparum (Pf) makes it difficult to limit infections and reduce the risk of severe malaria. Improved understanding of Pf blood-stage growth and development would provide new opportunities to evaluate and interfere with successful completion of the parasite's life cycle. Cultured blood stage Pf was incubated with Hoechst 33342 (HO) and thiazole orange (TO) to stain DNA and total nucleic acids, respectively. Correlated HO and TO fluorescence emissions were then measured by flow cytometry. Complex bivariate data patterns were analyzed by manual cluster gating to quantify parasite life cycle stages. The permutations of viable staining with both reagents were tested for optimal detection of parasitized RBC (pRBC). Pf cultures were exposed to HO and TO simultaneously to achieve optimal staining of pRBC and consistent quantification of early and late stages of the replicative cycle (rings through schizonts). Staining of Pf nucleic acids allows for analysis of parasite development in the absence of fixatives, lysis, or radioactivity to enable examination of erythrocytes from parasite invasion through schizont rupture using sensitive and rapid assay procedures. Investigation of the mechanisms by which anti-malarial drugs and antibodies act against different Pf lifecycle stages will be aided by this cytometric strategy.

A new biomarker for mitotic cells.

Many epitopes are phosphorylated during mitosis. These epitopes are useful biomarkers for mitotic cells. The most commonly used are MPM-2 and serine 10 of histone H3. Here we investigated the use of an antibody generated against a phospho peptide matching residues 774-788 of the human retinoblastoma protein 1 (Rb) to detect mitotic cells. Human cell lines were stained with DNA dyes and antibodies reactive with epitopes defined by antibody MPM-2, phospho-S10-histone-H3, and the phospho-serine peptide, TRPPTLSPIPHIPRC (phospho-S780-Rb). Immunoreactivity and DNA content were measured by flow and image cytometry. Correlation and pattern recognition analyses were performed on list mode data. Western blots and immunoprecipitation were used to investigate the number of peptides reactive with phospho-S780-Rb and the relationship between reactivity with this antibody and MPM-2. Costaining for bromodeoxyuridine (BrdU) was used to determine acid resistance of the phospho-S780-Rb epitope. Cell cycle related phospho-S780-Rb immunofluorescence correlated strongly with that of MPM-2. Laser scanning cytometry showed that phospho-S780-Rb immunofluorescence is expressed at high levels on all stages of mitotic cells. Western blotting and immunoprecipitation showed that the epitope is expressed on several peptides including Rb protein. Costaining of BrdU showed that the epitope is stable to acid. Kinetic experiments showed utility in complex cell cycle analysis aimed at measuring cell cycle transition state timing. The phospho-S780-Rb epitope is a robust marker of mitosis that allows cytometric detection of mitotic cells beginning with chromatin condensation and ending after cytokinesis. Costaining of cells with DNA dyes allows discrimination and counting of mitotic cells and post-cytokinetic ("newborn") cells. To facilitate use without confusion about specificity, we suggest the trivial name, pS780 for this mitotic epitope.

Dysregulated human myeloid nuclear differentiation antigen expression in myelodysplastic syndromes: evidence for a role in apoptosis.

Reduced levels of human myeloid nuclear differentiation antigen (MNDA) gene transcripts have been detected in both familial and sporadic cases of myelodysplastic syndromes (MDS). Numerous reports implicate elevated apoptosis/programmed cell death and death ligands and their receptors in the pathogenesis of MDS. MNDA and related proteins contain the pyrin domain that functions in signaling associated with programmed cell death and inflammation. We tested the hypothesis that MNDA is involved in the regulation of programmed cell death in human myeloid hematopoietic cells. Clones of K562 cells (MNDA-null) that expressed ectopic MNDA protein were established using retroviral transduction. MNDA-expressing K562 clones were resistant to tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-induced apoptosis, but were not protected from programmed cell death induced with genotoxic agents or H(2)O(2). MNDA protein expression assessed in control and intermediate and high-grade MDS marrows showed several patterns of aberrant reduced MNDA. These variable patterns of dysregulated MNDA expression may relate to the variable pathophysiology of MDS. We propose that MNDA has a role regulating programmed cell death in myeloid progenitor cells, and that its down-regulation in MDS is related to granulocyte-macrophage progenitor cell sensitivity to TRAIL-induced programmed cell death.

Multiparameter Cell Cycle Analysis.

Cell cycle cytometry and analysis are essential tools for studying cells of model organisms and natural populations (e.g., bone marrow). Methods have not changed much for many years. The simplest and most common protocol is DNA content analysis, which is extensively published and reviewed. The next most common protocol, 5-bromo-2-deoxyuridine S phase labeling detected by specific antibodies, is also well published and reviewed. More recently, S phase labeling using 5'-ethynyl-2'-deoxyuridine incorporation and a chemical reaction to label substituted DNA has been established as a basic, reliable protocol. Multiple antibody labeling to detect epitopes on cell cycle regulated proteins, which is what this chapter is about, is the most complex of these cytometric cell cycle assays, requiring knowledge of the chemistry of fixation, the biochemistry of antibody-antigen reactions, and spectral compensation. However, because this knowledge is relatively well presented methodologically in many papers and reviews, this chapter will present a minimal Methods section for one mammalian cell type and an extended Notes section, focusing on aspects that are problematic or not well described in the literature. Most of the presented work involves how to segment the data to produce a complete, progressive, and compartmentalized cell cycle analysis from early G1 to late mitosis (telophase). A more recent development, using fluorescent proteins fused with proteins or peptides that are degraded by ubiquitination during specific periods of the cell cycle, termed "Fucci" (fluorescent, ubiquitination-based cell cycle indicators) provide an analysis similar in concept to multiple antibody labeling, except in this case cells can be analyzed while living and transgenic organisms can be created to perform cell cycle analysis ex or in vivo (Sakaue-Sawano et al., Cell 132:487-498, 2007). This technology will not be discussed.

p53 negatively regulates intestinal immunity by delaying mucosal T cell cycling.

To mount an effective immune response, T cells must divide in response to antigen contact. To maintain tolerance, mucosal lamina propria T cells (LPTs) may adapt their cycling to an antigen-rich gut stimulatory environment. Here, we compared the cell cycle kinetics of LPTs and peripheral blood T cells (PBTs) before and after CD3- and CD2-mediated activation. While CD3-activated naive (CD45RA(+)) and memory (CD45RO(+)) PBTs peaked in the S and G2/M phase at 2-3 days, CD3-activated LPTs peaked at 4-6 days. In contrast, CD2 activation induced modest PBT but vigorous LPT cycling. The doubling time of CD3-activated PBTs was 1 day, while that of CD3- or CD2-activated LPTs was 2 days. LPTs failed to upregulate cyclin-dependent kinase 4 and cyclin D3, but Rb phosphorylation and cyclin A and B1 upregulation were induced by CD2 engagement. The extents of clonal expansion in LPT and PBT were comparable, indicating that LPTs' slow replication delays but does not hinder cell division. CD2-activated LPTs displayed a striking upregulation of p53, whose blockade by antisense oligonucleotides accelerated their S phase transit time to that of CD3-activated PBTs. By slowing LPT cycling, p53 may act as a negative regulator of mucosal immunity, promoting immunological tolerance by preventing excessive T cell replication.

Phase I and correlative study of combination bryostatin 1 and vincristine in relapsed B-cell malignancies.

PURPOSE: Bryostatin 1 activates protein kinase C (PKC) with short-term exposure and results in depletion of PKC with prolonged exposure. Preclinical in vitro and in vivo studies demonstrate synergistic activity and increased tumor apoptosis in B-cell malignancies when a prolonged infusion of bryostatin 1 is followed by vincristine. EXPERIMENTAL DESIGN: We embarked on a Phase I trial of bryostatin 1 as a 24-h continuous infusion followed by bolus vincristine in patients with refractory B-cell malignancies other than acute leukemias. Twenty-four evaluable patients were enrolled. RESULTS: The dose-limiting toxicity was myalgia. The MTD and recommended Phase II dose of bryostatin 1 was 50 microg/m2/24 h followed by vincristine 1.4 mg/m2 (maximum total dose of 2 mg) repeated every 2 weeks. Significant antitumor activity was observed in this relapsed population, including patients who had failed high-dose chemotherapy. This included 5 durable complete and partial responses and 5 patients with stable disease lasting > or =6 months (range, 6-48+ months). Median time to response was 8 months. Correlative studies demonstrated a progressive increase in serum interleukin-6 with bryostatin 1 infusion followed by an additional increase after vincristine. Flow cytometry for detection of apoptosis in B and T cells showed an initial decrease in apoptotic frequency in CD5+ cells within 6 h of bryostatin 1 infusion compatible with its known increase in PKC activity in the majority of patients followed by a return to baseline or overall increase in apoptotic frequency after completion of infusion. All (5 of 5) patients who had an overall increase in apoptotic frequency in CD5+ cells achieved either a clinical response or prolonged stable disease. Four of these 5 patients did not have the initial decrement in apoptosis at 6 h. CONCLUSIONS: Given the lack of myelosuppression and early evidence of clinical efficacy, additional exploration of this regimen in non-Hodgkin's lymphoma and multiple myeloma is warranted.