A phase II multicenter trial of hyperCVAD MTX/Ara-C and rituximab in patients with previously untreated mantle cell lymphoma; SWOG 0213.

BACKGROUND: Rituximab-hyper-CVAD alternating with rituximab-high-dose methotrexate and cytarabine is a commonly utilized regimen in the United States for mantle cell lymphoma (MCL) based on phase II single institutional data. To confirm the clinical efficacy of this regimen and determine its feasibility in a multicenter study that includes both academic and community-based practices, a phase II study of this regimen was conducted by SWOG. PATIENTS AND METHODS: Forty-nine patients with advanced stage, previously untreated MCL were eligible. The median age was 57.4 years (35-69.8 years). RESULTS: Nineteen patients (39%) did not complete the full scheduled course of treatment due to toxicity. There was one treatment-related death and two cases of secondary myelodysplastic syndrome (MDS). There were 10 episodes of grade 3 febrile neutropenia, 19 episodes of grade 3 and 1 episode of grade 4 infection. With a median follow-up of 4.8 years, the median progression-free survival was 4.8 years (5.5 years for those ≤ 65 years) and the median overall survival (OS) was 6.8 years. CONCLUSIONS: Although this regimen is toxic, it is active for patients ≤ 65 years of age and can be given both at academic centers and in experienced community centers.

Efficient modification of human chromosomal alleles using recombination-proficient chicken/human microcell hybrids.

Targeted modification of human chromosomal alleles by homologous recombination is a powerful approach to study gene function, but gene targeting in mammalian cells is an inefficient process. In contrast, gene targeting in a chicken pre-B cell line, DT40, is highly efficient. We have transferred human chromosome 11 into DT40 cells by microcell fusion, and find that the resulting hybrids are recombination-proficient. In these cells, targeting efficiencies into the chicken ovalbumin locus were > 90% and into the human beta-globin and Ha-ras loci were 10-15%. These modified human chromosomes can be transferred subsequently to mammalian cells for functional tests. This chromosome shuttle system allows for the efficient homologous modification of human chromosomal genes, and for subsequent phenotypic analyses of the modified alleles in different mammalian cell types.

Bortezomib in patients with relapsed or refractory mantle cell lymphoma: updated time-to-event analyses of the multicenter phase 2 PINNACLE study.

BACKGROUND: We previously reported results of the phase 2, multicenter PINNACLE study, which confirmed the substantial single-agent activity of bortezomib in patients with relapsed or refractory mantle cell lymphoma (MCL). MATERIALS AND METHODS: We report updated time-to-event data, in all patients and by response to treatment, after extended follow-up (median 26.4 months). RESULTS: Median time to progression (TTP) was 6.7 months. Median time to next therapy (TTNT) was 7.4 months. Median overall survival (OS) was 23.5 months. In responding patients, median TTP was 12.4 months, median duration of response (DOR) was 9.2 months, median TTNT was 14.3 months, and median OS was 35.4 months. Patients achieving complete response had heterogeneous disease characteristics; among these patients, median TTP and DOR were not reached, and median OS was 36.0 months. One-year survival rate was 69% overall and 91% in responding patients. Median OS from diagnosis was 61.1 months, after median follow-up of 63.7 months. Activity was seen in patients with refractory disease and patients relapsing following high-intensity treatment. Toxicity was generally manageable. CONCLUSIONS: Single-agent bortezomib is associated with lengthy responses and notable survival in patients with relapsed or refractory MCL, with considerable TTP and TTNT in responding patients, suggesting substantial clinical benefit.

Participation of the human beta-globin locus control region in initiation of DNA replication.

The human beta-globin locus control region (LCR) controls the transcription, chromatin structure, and replication timing of the entire locus. DNA replication was found to initiate in a transcription-independent manner within a region located 50 kilobases downstream of the LCR in human, mouse, and chicken cells containing the entire human beta-globin locus. However, DNA replication did not initiate within a deletion mutant locus lacking the sequences that encompass the LCR. This mutant locus replicated in the 3' to 5' direction. Thus, interactions between distantly separated sequences can be required for replication initiation, and factors mediating this interaction appear to be conserved in evolution.

The locus control region is necessary for gene expression in the human beta-globin locus but not the maintenance of an open chromatin structure in erythroid cells.

Studies in many systems have led to the model that the human beta-globin locus control region (LCR) regulates the transcription, chromatin structure, and replication properties of the beta-globin locus. However the precise mechanisms of this regulation are unknown. We have developed strategies to use homologous recombination in a tissue culture system to examine how the LCR regulates the locus in its natural chromosomal environment. Our results show that when the functional components of the LCR, as defined by transfection and transgenic studies, are deleted from the endogenous beta-globin locus in an erythroid background, transcription of all beta-globin genes is abolished in every cell. However, formation of the remaining hypersensitive site(s) of the LCR and the presence of a DNase I-sensitive structure of the beta-globin locus are not affected by the deletion. In contrast, deletion of 5'HS5 of the LCR, which has been suggested to serve as an insulator, has only a minor effect on beta-globin transcription and does not influence the chromatin structure of the locus. These results show that the LCR as currently defined is not necessary to keep the locus in an "open" conformation in erythroid cells and that even in an erythroid environment an open locus is not sufficient to permit transcription of the beta-like globin genes.

Analysis of mice containing a targeted deletion of beta-globin locus control region 5' hypersensitive site 3.

To examine the function of murine beta-globin locus region (LCR) 5' hypersensitive site 3 (HS3) in its native chromosomal context, we deleted this site from the mouse germ line by using homologous recombination techniques. Previous experiments with human 5' HS3 in transgenic models suggested that this site independently contains at least 50% of total LCR activity and that it interacts preferentially with the human gamma-globin genes in embryonic erythroid cells. However, in this study, we demonstrate that deletion of murine 5' HS3 reduces expression of the linked embryonic epsilon y- and beta H 1-globin genes only minimally in yolk sac-derived erythroid cells and reduces output of the linked adult beta (beta major plus beta minor) globin genes by approximately 30% in adult erythrocytes. When the selectable marker PGK-neo cassette was left within the HS3 region of the LCR, a much more severe phenotype was observed at all developmental stages, suggesting that PGK-neo interferes with LCR activity when it is retained within the LCR. Collectively, these results suggest that murine 5' HS3 is not required for globin gene switching; importantly, however, it is required for approximately 30% of the total LCR activity associated with adult beta-globin gene expression in adult erythrocytes.

Long-distance control of origin choice and replication timing in the human beta-globin locus are independent of the locus control region.

DNA replication in the human beta-globin locus is subject to long-distance regulation. In murine and human erythroid cells, the human locus replicates in early S phase from a bidirectional origin located near the beta-globin gene. This Hispanic thalassemia deletion removes regulatory sequences located over 52 kb from the origin, resulting in replication of the locus from a different origin, a shift in replication timing to late S phase, adoption of a closed chromatin conformation, and silencing of globin gene expression in murine erythroid cells. The sequences deleted include nuclease-hypersensitive sites 2 to 5 (5'HS2-5) of the locus control region (LCR) plus an additional 27-kb upstream region. We tested a targeted deletion of 5'HS2-5 in the normal chromosomal context of the human beta-globin locus to determine the role of these elements in replication origin choice and replication timing. We demonstrate that the 5'HS2-5-deleted locus initiates replication at the appropriate origin and with normal timing in murine erythroid cells, and therefore we conclude that 5'HS2-5 in the classically defined LCR do not control replication in the human beta-globin locus. Recent studies also show that targeted deletion of 5'HS2-5 results in a locus that lacks globin gene expression yet retains an open chromatin conformation. Thus, the replication timing of the locus is closely correlated with nuclease sensitivity but not globin gene expression.

Hematopoietic cell transplantation from HLA-identical sibling donors after low-dose radiation-based conditioning for treatment of CML.

A total of 24 patients (median age 58; range, 27-71 years) with chronic myeloid leukemia (CML) in first chronic (CP1) (n=14), second chronic (n=4), or accelerated phase (n=6) who were not candidates for conventional hematopoietic cell transplantation (HCT), received nonmyeloablative HCT from HLA-matched siblings a median of 28.5 (range, 11-271) months after diagnosis. They were conditioned with 2 Gy total body irradiation (TBI) alone (n=8) or combined with fludarabine, 90 mg/m(2) (n=16). Postgrafting immunosuppression included cyclosporine and mycophenolate mofetil. All patients initially engrafted. However, 4 of 8 patients not given fludarabine experienced nonfatal rejection while all others had sustained engraftment. With a median follow-up of 36 (range, 4-49) months, 13 of 24 patients (54%) were alive and in complete remission. There were five (21%) deaths from nonrelapse mortality, one (4%) during the first 100 days after transplant. The proportions of grade II, III, and IV acute GVHD were 38, 4, and 8%, respectively. The 2-year estimate of chronic GVHD was 32%. The 2-year survival estimates for patients in CP1 (n=14) and beyond CP1 (n=10) were 70 and 56%, respectively. This study shows encouraging remission rates for patients with CML not eligible for conventional allografting.

Reduced beta-globin gene expression in adult mice containing deletions of locus control region 5' HS-2 or 5' HS-3.

To gain insights into the functions of individual DNA'se hypersensitive sites within the beta globin locus control region (LCR), we deleted the endogenous 5' HS-2 and HS-3 regions from the mouse germline using homologous recombination techniques. We demonstrated that the deletion of either murine 5' HS-2 or 5' HS-3 reduced the expression of the embryonic epsilon y and beta h1 globin genes minimally in yolk sac-derived erythrocytes, but that both knockouts reduced the output of the adult beta (beta-Major + beta-Minor) globin genes by approximately 30% in adult erythrocytes. When the selectable marker PGK-Neo cassette was retained within either the HS-2 or HS-3 region, a much more severe reduction in globin gene expression was observed at all developmental stages. PGK-Neo was shown to be expressed in an erythroid-specific fashion when it was retained in the HS-3 position. These results show that neither 5' HS-2 nor HS-3 is required for the activity of embryonic globin genes, nor are these sites required for correct developmental switching. However, each site is required for approximately 30% of the total LCR activity associated with adult beta-globin gene expression in adult red blood cells. Each site therefore contains some non-redundant information that contributes to adult globin gene function.

G-CSF mobilized vs conventional donor lymphocytes for therapy of relapse or incomplete engraftment after allogeneic hematopoietic transplantation.

There is little data comparing the activity and toxicity of donor lymphocyte therapy with granulocyte (G)-CSF-mobilized cells (G-donor lymphocyte infusion (DLI)) with the conventionally collected DLI (C-DLI) after allogeneic blood or marrow transplantation. We retrospectively evaluated 67 patients to compare the efficacy and toxicity of GCSF-mobilized DLI with C-DLI in the treatment of relapse of malignant disease or poor donor engraftment post transplant. We assessed clinical outcomes that may represent the immunological outcome of DLI. The median OS was 210 days (range 3-2436 days), 291 days (range 17-1491 days) in the G-DLI group (15 patients) and 207.5 days (range 3-2436 days) in the C-DLI group (52 patients). The median PFS time was 72 days (range 8-1491 days) in the G-DLI group vs 82 days (range 1-2436 days) in the C-DLI group. Rates of post DLI GVHD and improvement in donor engraftment were similar in the G-DLI and C-DLI groups. We conclude that G-DLI appears to have similar therapeutic activity to that seen with C-DLI, and where such cells are available they may be substituted for conventional donor lymphocytes.

Replication of alpha and beta globin DNA sequences occurs during early S phase in murine erythroleukemia cells.

Murine erythroleukemia cells (MELC) can be induced to express the characteristics of erythroid differentiation by a variety of agents. Previous studies indicate that an action of inducer, occurring during early S phase, may be critical to the expression of differentiated characteristics such as initiation of accumulation of newly synthesized alpha and beta globin mRNAs. In this investigation, the time of replication of globin genes in MELC was studied. DNA was isolated from synchronous populations of cells obtained by centrifugal elutriation. Newly replicated DNA sequences were prepared from synchronized cells cultured for 1 1/2 hr with 5-bromodeoxyuridine; bromodeoxyuridine-containing DNA was isolated by CsCl gradient centrifugation. By employing cloned probes for hybridization to newly synthesized DNA, it was found that alpha and beta globin gene sequences are replicated early in S phase, while ribosomal RNA gene sequences are replicated to about the same extent in early, middle, and late S phases.

Asynchronous DNA replication within the human beta-globin gene locus.

The timing of DNA replication of the human beta-globin gene locus has been studied by blot hybridization of newly synthesized BrdUrd-substituted DNA from cells in different stages of the S phase. Using probes that span greater than 120 kilobases across the human beta-globin gene locus, we show that the majority of this domain replicates in early S phase in the human erythroleukemia cell line K562 and in middle-to-late S phase in the lymphoid cell line Manca. However, in K562 cells three small regions display a strikingly different replication pattern than adjacent sequences. These islands, located in the inter-gamma-globin gene region and approximately 20 kilobases 5' to the epsilon-globin gene and 20 kilobases 3' to the beta-globin gene, replicate later and throughout S phase. A similar area is also present in the alpha-globin gene region in K562 cells. We suggest that these regions may represent sites of termination of replication forks.

Role of polyadenylic acid in a deoxyribonucleic acid-membrane fraction extracted from pneumococci.

After the addition of radioactive polyadenylic acid to cell suspensions of pneumocci, part of the radioactivity becomes associated with a deoxyribonucleic acid (DNA)-membrane fraction extracted from the cells. A variety of techniques show that a portion of this associated radioactivity may represent oligoadenylates complexed to DNA, probaby as part of a ribonucleic acid (RNA) component. Polyadenylic acid, which had previously been shown to enhance DNA synthesis in cell suspensions (Firshein and Benson, 1968), also enhances the extent of DNA synthesis by the DNA-membrane fraction in vitro under specific conditions of concentration and conformation. The mechanism of action of this enhancement may be related to the ability of oligoadenylates to increase the number of initiation sites for DNA replication by stimulating the production of an RNA primer, thus providing additional 3'-OH groups with which DNA polymerase can react.

An "in-out" strategy using gene targeting and FLP recombinase for the functional dissection of complex DNA regulatory elements: analysis of the beta-globin locus control region.

The human beta-globin locus control region (LCR) is a complex DNA regulatory element that controls the expression of the cis-linked beta-like globin genes located in the 55 kilobases 3' of the LCR. We have initiated the functional analysis of the LCR by homologous recombination in murine erythroleukemia cell somatic hybrids that carry a single copy of human chromosome 11 on which the beta-globin locus is situated. High-level expression of the human beta-globin gene normally occurs when these hybrid cells are induced to differentiate. We have reported that the insertion of an expressed selectable marker gene (driven by the Friend virus enhancer/promoter) into the LCR disrupts the LCR-mediated regulation of globin transcription. In these cells, beta-globin is no longer expressed when the cells differentiate; instead, expression of the selectable marker gene increases significantly after differentiation. Since present techniques for homologous recombination require the insertion of a selectable marker, further progress in using homologous recombination to analyze the LCR depends on deletion of the selectable marker and demonstration that the locus functions normally after the insertion, expression, and deletion of the selectable marker. Here we show that after precise deletion of the selectable marker by using the FLP recombinase/FRT (FLP recombinase target) system, the locus functions as it did before the homologous recombination event. These studies demonstrate the feasibility of using homologous recombination to analyze the LCR in particular, and other complex cis-regulatory DNA elements in general, in their normal chromosomal context.

Inactivation of the human beta-globin gene by targeted insertion into the beta-globin locus control region.

The human beta-globin locus control region (LCR) is a complex regulatory element that controls the erythroid-specific expression of all cis-linked globin genes. The LCR is composed of five DNase I hypersensitive sites (HS) spanning 16 kb and located greater than 50 kb upstream of the beta-globin gene on chromosome 11. Constructs containing all or some of these HS have been shown to produce high-level erythroid-specific expression of linked genes in transgenic mice and transfected cells. In all transgenic and transfection experiments reported to date, however, the spatial relationships between the LCR and globin genes have been disrupted. We have used homologous recombination (HR) as an approach to gain insights into the potential interactions between the LCR and globin genes in their native locations. A hygromycin B resistance (hygro(R)) gene was inserted into the human beta-globin LCR on chromosome 11 in a mouse/human hybrid erythroid cell line that expresses the human beta-globin gene after the induction of differentiation. As a consequence of this targeted insertion, the beta-globin gene is transcriptionally inactive and not inducible. In contrast, the hygro(R) gene within the LCR is inducible, whereas randomly integrated hygro(R) genes are not inducible in these cells. The chromatin structure of the targeted locus is also altered. A new DNase I HS is present in the enhancer/promoter of the hygro(R) gene inserted into the LCR, whereas a HS normally present in the LCR 3' to the insertion is lost and the beta-globin gene promoter HS is not detectable. These results are consistent with the promoter/enhancer competition model for LCR function and globin gene switching.

Description and targeted deletion of 5' hypersensitive site 5 and 6 of the mouse beta-globin locus control region.

The most upstream hypersensitive site (HS) of the beta-globin locus control region (LCR) in humans (5' HS 5) and chickens (5' HS 4) can act as an insulating element in some gain of function assays and may demarcate a beta-globin domain. We have mapped the most upstream HSs of the mouse beta-globin LCR and sequenced this region. We find that mice have a region homologous to human 5' HS 5 that is associated with a minor HS. In addition we map a unique HS upstream of 5' HS 5 and refer to this novel site as mouse 5' HS 6. We have also generated mice containing a targeted deletion of the region containing 5' HS 5 and 6. We find that after excision of the selectable marker in vivo, deletion of 5' HS 5 and 6 has a minimal effect on transcription and does not prevent formation of the remaining LCR HSs. Taken together these findings suggest that the most upstream HSs of the mouse beta-globin LCR are not necessary for maintaining the beta-globin locus in an active configuration or to protect it from a surrounding repressive chromatin environment.

Induction of murine erythroleukemia differentiation by actinomycin D.

Murine erythroleukemia cells are induced to differentiate by 0.5-5 ng of actinomycin D per ml. Murine erythroleukemia cells cultured with actinomycin D prolong cell doubling time but achieve the same density after 5 days as cells without inducer. Actinomycin D causes over 95% of the cells to become benzidine-reactive. [(3)H]Actinomycin D uptake into DNA can be detected within 2 hr and reaches a maximum (approximately 0.1 pmol/10(6) cells) by 10-12 hr. It is estimated that about one out of 10(5) dG.dC pairs is bound to actinomycin D. Commitment to differentiation, assayed by transfer of cells to culture without inducer, was detected as early as 5 hr. Unlike Me(2)SO, which causes a transient prolongation in G(1) at about 15-20 hr, cells cultured with actinomycin D show a more sustained increase in the proportion of the cells in G(1). Globin mRNA accumulation was detectable by 19 hr in culture. Alteration in DNA stability in alkaline sucrose gradients was detected by 19 hr. Actinomycin D induces synthesis of Hb(maj) and Hb(min) in approximately equal amounts. A decrease in rates of synthesis of RNA, DNA, and total protein occurs in cells cultured with actinomycin D, as well as in cells cultured with Me(2)SO. No evidence for an early action of actinomycin D at the plasma membrane was obtained by measurement of changes in cell volume or (86)RbCl uptake. Taken together, the present results indicate that actinomycin D is a potent inducer of differentiation of murine erythroleukemia cells and suggest that the target of its effect may be at the level of DNA.

A deletion of the human beta-globin locus activation region causes a major alteration in chromatin structure and replication across the entire beta-globin locus.

Naturally occurring deletions that remove sequences located approximately 60 kb upstream of the human adult beta-globin gene result in the failure to transcriptionally activate the cis-linked globin genes in erythroid cells. In addition, transfection, transgenic, and somatic cell hybrid studies have revealed that sequences within this region are essential for the developmentally regulated high-level expression of cis-linked globin genes. This regulatory region located at the 5' end of the beta-globin locus has been termed the locus activation region (LAR). Using somatic cell hybrids, we have studied the chromatin structure and timing of DNA replication of the normal human beta-globin locus and a locus containing a de novo 25-kb deletion that removes elements of the LAR. As a result of this deletion, the entire beta-globin locus and sequences approximately 100 kb 5' and 3' of the adult beta-globin gene are DNase I-resistant and do not form characteristic distant hypersensitive sites. These sequences also replicate late in S phase in an erythroid cell background. In contrast, the sequences of the normal locus are DNase I sensitive and early replicating. These results suggest that the LAR is required for both the erythroid-specific chromatin structure and timing of DNA replication over a large physical distance.