Comparison and modelling of pegylated or unpegylated G-CSF schedules in CHOP-14 regimen of elderly patients with aggressive B-cell lymphoma.

Bi-weekly (R)-CHOP therapy is one of the standard treatmentS for elderly patients with aggressive B-cell lymphoma, but it is only feasible with supportive G-CSF treatment. In the trials of the DSHNHL, either unpegylated G-CSF was given daily over 7 or 10 days or pegylated G-CSF was applied at day 4 of each cycle. These schedules were planned on the basis of simulations of a biomathematical pharmacokinetic/pharmacodynamic model. By analysing the observed data, we investigated whether our model predictions were correct and whether even better schedules can be proposed. We used data on 249 matched patients of two prospective trials, RICOVER-60 and PEGFILGRASTIM. The three G-CSF-schedules showed similar outcomes regarding leukocytopenia, infections and days in hospital, with pegylated G-CSF having slightly but not significantly better scores in all three endpoints. Regarding pegylated G-CSF, the best timing is predicted to be any day between days 4 and 7. With respect to unpegylated G-CSF, the starting day is less important, but it should be continued until the end of each cycle.The three G-CSF-schedules are interchangeable in (R)-CHOP-14 for elderly patients with aggressive B-cell lymphoma. Our model correctly predicts time courses of leukocytes. Further model predictions are presented, which can be tested in subsequent clinical trials.

FXa-induced responses in vascular wall cells are PAR-mediated and inhibited by ZK-807834.

During thrombosis, vascular wall cells are exposed to clotting factors, including the procoagulant proteases thrombin and factor Xa (FXa), both known to induce cell signaling. FXa shows dose-dependent induction of intracellular Ca(2+) transients in vascular wall cells that is active-site-dependent, Gla-domain-independent, and enhanced by FXa assembly into the prothrombinase complex. FXa signaling is independent of prothrombin activation as shown by the lack of inhibition by argatroban, hirudin and the sulfated C-terminal peptide of hirudin (Hir(54-65)(SO3(-))). This peptide binds to both proexosite I in prothrombin and exosite I in thrombin. In contrast, signaling is completely blocked by the FXa inhibitor ZK-807834 (CI-1031). No inhibition is observed by peptides which block interaction of FXa with effector cell protease 1 receptor (EPR-1), indicating that this receptor does not mediate signaling in the cells assayed. Receptor desensitization studies with thrombin or peptide agonists (PAR-1 or PAR-2) and experiments with PAR-1-blocking antibodies indicate that signaling by FXa is mediated by both PAR-1 and PAR-2. Potential pathophysiological responses to FXa include increased cell proliferation, increased production of the proinflammatory cytokine IL-6 and increased production of prothrombotic tissue factor. These cellular responses, which may complicate vascular disease, are inhibited by ZK-807834.

Embryonic expression of the Gax homeodomain protein in cardiac, smooth, and skeletal muscle.

Gax is a homeobox-containing gene that has been detected in adult cardiovascular tissues and exhibits a growth arrest-specific pattern of expression in cultured vascular myocytes. To study the regulation of gax during development, we performed immunohistochemistry and in situ hybridization on mouse embryos. Gax was present in mesodermally and, as with other homeobox genes, neuroectodermally derived tissues. Early mesodermal protein expression was limited to the lateral plate and somitic mesoderm. Gax in the cardiac muscle lineage exhibited a biphasic pattern of expression. Expression was prominent in the heart tube of the earliest cardiomyocytes and remained prominent through the looping stage (day 12.5 post coitum [pc]) but fell below the threshold of detection in atria and ventricles by day 13.5 pc. At day 15.5 pc, Gax protein was again detectable but restricted to cells within the compact layer of the ventricular myocardium. Gax expression was also noted in smooth muscle cells as early as day 9.5 pc. In the skeletal muscle lineage, Gax protein was expressed at the onset of somitogenesis before the expression of the myogenic basic helix-loop-helix and MEF2/RSRF family proteins. Subsequently, it was noted at day 9.5 pc in premyogenic cells migrating into head, trunk, and limb buds. Gax was detected in myotomes, premuscle masses, and mature muscle groups. These data suggest an important developmental role for Gax in all muscle lineages.

Inactivation of a c-Myb/estrogen receptor fusion protein in transformed primary cells leads to granulocyte/macrophage differentiation and down regulation of c-kit but not c-myc or cdc2.

Primary murine fetal hemopoietic cells were transformed with a fusion protein consisting of the ligand-binding domain of the estrogen receptor and a carboxyl-terminally truncated c-Myb protein (ERMYB). The ERMYB-transformed hemopoietic cells exhibit an immature myeloid phenotype when grown in the presence of beta-estradiol. Upon removal of beta-estradiol, the ERMYB cells display increased adherence, decreased clonogenicity and differentiate to cells exhibiting granulocyte or macrophage morphology. The expression of the c-myc, c-kit, cdc2 and bcl-2 genes, which are putatively regulated by Myb, was investigated in ERMYB cells grown in the presence or absence of beta-estradiol. Neither c-myc nor cdc2 expression was down-regulated after removal of beta-estradiol demonstrating that differentiation is not a consequence of decreased transactivation of these genes by ERMYB. While bcl-2 expression was reduced by 50% in ERMYB cells grown in the absence of beta-estradiol, there was no increase in DNA laddering, suggesting that Myb was not protecting ERMYB cells from apoptosis. In contrast, a substantial (200-fold) decrease in c-kit mRNA level was observed following differentiation of ERMYB cells, and c-kit mRNA could be partially re-induced by the re-addition of beta-estradiol. Furthermore, a reporter construct containing the c-kit promoter was activated when cotransfected with a Myb expression vector, providing further evidence of a role for Myb in the regulation of c-kit.

The MYC protein activates transcription of the alpha-prothymosin gene.

The proto-oncogene MYC encodes a nuclear protein whose biochemical and physiological functions remain uncertain. We used an estrogen-regulated version of the MYC protein to explore these functions. Activation of MYC in quiescent rat and mouse fibroblasts elicited re-entry into and progression through the cell cycle, bypassing early events that would follow stimulation of the cells with serum. Activation of MYC led to a rapid increase in transcription of the alpha-prothymosin gene, even in the absence of protein synthesis. We conclude that the product of MYC acts directly on transcription, in accord with inferences based on the structure of the MYC protein. The function of alpha-prothymosin is not known, but our results suggest that the protein may play a role in the proliferation of mammalian cells.

Pseudorabies virus immediate-early gene overlaps with an oppositely oriented open reading frame: characterization of their promoter and enhancer regions.

The immediate-early (IE) gene of pseudorabies virus (PRV) has recently been sequenced for two virus strains. To investigate IE gene regulation and to examine the genome segment reported to encode latency-related transcripts in opposite polarity to the IE gene, sequence analysis has been extended by 5 kb from each end of the IE gene. The IE promoter (P1) was found to be more complex than previously recognized: it consisted of nine imperfect repeats, each containing five to six different consensus elements for transcription factor binding. A second promoter (P2) was discovered downstream of the IE gene. It contained numerous octamer consensus sequences (ATGCAAAT) and recognition sites for transcription factor Sp1; specific binding of nuclear proteins to four Sp1 sites was detected. An open reading frame (ORF3) bordering on P2 was identified, oriented antiparallel to the IE gene. Potential enhancer elements (E3 and E4) were isolated by the enhancer trap technique. Linked to P1 and a CAT indicator gene, E3 acted as an enhancer and E4 as a silencer. The PRV IE gene product repressed transcription from its own promoter and activated the SV40 early promoter. The transactivating virion protein Vmw65 of HSV1 had an opposite effect on these promoters.

A temperature-sensitive phenotype of avian myeloblastosis virus: determinants that influence the production of viral mRNAs.

The oncogene v-myb of avian myeloblastosis virus is expressed from an mRNA that arises by splicing of the viral genome. In previous work, we described a mutant strain of avian myeloblastosis virus (tsAMV) that elicits temperature-sensitive transformation and suggested that the mutation affects production of the mRNA for v-myb. We now report that the principal determinant of the biochemical phenotype of tsAMV is a point mutation located in a crucial region of the splice acceptor site for v-myb mRNA. The mutation reduces v-myb mRNA production but could account for the conditional phenotype only in combination with an independent effect of temperature on the splicing of both wild-type and mutant viral RNAs, which we also describe here. Our findings dramatize the manner in which retroviruses normally control the splicing of their RNAs and implicate the sequence of the splice acceptor site in the control.

Redundancy of information in enhancers as a principle of mammalian transcription control.

In contrast to prokaryotes, in which strong transcriptional signals can be located within very short DNA segments, typical mammalian enhancers are about 200 base-pairs long. We reasoned that a minimal length of enhancer-active DNA is required for a high transcription rate in higher eukaryotes, and that segments from a single enhancer or from different enhancers might be multimerized or combined to satisfy such a requirement. To test this, enhancer fragments from different viruses were joined in a recombinant simian virus 40 (SV40) and screened for efficiency of viral growth. The 48 combinations tested show that the hypothesis is basically correct. For example, two subfunctional heterologous enhancer fragments can together form a functional enhancer. No enhancer shorter than 84 base-pairs could promote SV40 growth, i.e. in no case did we find a short "superstrong" enhancer segment. To test whether multimerization of a short fragment would result in a strong enhancer, we have synthesized a 50 base-pair enhancer segment derived from Herpesvirus saimiri. One to six copies of this oligonucleotide gave an incremental increase in enhancer activity. We propose, therefore, that mammalian gene regulation is based on a redundancy of information that can be provided either by a combination of different DNA sequence elements, or by multiple copies of the same element. Also, the finding of strong and weak enhancers suggests that in most cases an enhancer is permanently required for transcription of a gene, rather than acting in an all-or-none fashion to establish a transcription complex, after which it becomes dispensable.

The SV40 enhancer can be dissected into multiple segments, each with a different cell type specificity.

The SV40 enhancer is known to be active in a wide variety of tissues and species. It contains a number of sequence motifs that can be bound by protein factors and whose integrity is essential for full enhancer activity. We have individually analyzed three synthetic oligonucleotides derived from sequences present within the SV40 enhancer: two oligonucleotides contain variants of the enhancer "core" sequence (designated corePVUII and coreC) and the third represents a region containing a decanucleotide homology to the immunoglobulin promoters/enhancers (designated SPHI). The oligonucleotides were multimerized and linked to a beta-globin test gene. Transcripts of the test gene were analyzed following transient expression in 10 cell lines representing a broad spectrum of tissues. We show that each of the three short segments can individually act as an enhancer when present in multiple copies. None of these enhancers is ubiquitously active; however, each shows activity in a distinctive subpopulation of cell lines. This cell type specificity is most remarkable in the case of the two oligonucleotide segments containing the core sequences. One of these is primarily active in CV-1 cells, whereas the other exhibits a cell type specificity identical to that of the entire enhancer, possibly identifying it as the most important sequence element within the native SV40 enhancer. Our data suggest that a particular cell type specificity is typical for individual enhancer segments, and that enhancers of differing specificity can be assembled from the individual sequence motifs by combining them in different patterns.

A transcription enhancer in the Herpesvirus saimiri genome.

Herpesvirus saimiri, an oncogenic agent of New World primates, has a linear double-stranded DNA genome of approximately 155 kb. To test its genome for the presence of a transcription enhancer, we have mixed randomly fragmented H. saimiri DNA with non-infectious, linear SV40 DNA lacking the 72-bp repeat enhancer region (the so-called SV40 enhancer trap) and co-transfected this DNA mixture into monkey CV-1 cells. Viable SV40-like viruses were generated by intracellular ligation/repair processes with short H. saimiri DNA fragments. One recombinant, SVHS-2, had integrated a 377-bp enhancer segment from the righthand region of the H. saimiri genome, 7 kb upstream of DNA sequences encoding an immediate-early mRNA. This enhancer sequence is contained within the non-repetitive portions of the viral genome known to be preserved episomally in all lymphoid tumor cell lines. Further recombinant viruses (SVHS-14, SVHS-7, and SVHS-8) essentially contain subsets of the 377-bp insert. Unlike in the previous enhancer trap experiments, where heterologous enhancers were incorporated without any sequence alterations, SVHS-14 and SVHS-7 have suffered short internal deletions of a very similar segment of the H. saimiri insert. This renders the enhancer more active, implying that the deleted segment, while it may have a role in the herpesvirus infection cycle, exerts a negative effect within the isolated enhancer.

Herpesvirus saimiri-induced lymphoblastoid rabbit cell line: growth characteristics, virus persistence, and oncogenic properties.

A nonproducer lymphoblastoid cell line (7710) containing the herpesvirus saimiri (HVS) genome was established from the HVS-positive spleen of a male, inbred New Zealand White rabbit (III/J strain) which had developed a well-differentiated lymphoma after inoculation of HVS and 12-O-tetradecanoylphorbol-13-acetate (TPA). Antibodies to HVS early and late antigens were detected in the serum of rabbit 7710 by indirect immunofluorescence and immunoprecipitation. The cell line was of T-cell origin, did not produce HVS, and could not be superinfected with HVS. However, HVS early antigens could be induced in the cells with n-butyric acid and TPA or TPA alone. On the other hand, late antigens were never observed, and infectious virus could not be rescued by cocultivation of 7710 cell with OMK cells. The 7710 cells were T-cell growth factor dependent, even after many in vitro passages. The 7710 cell line contained multiple copies of a nonintegrated, covalently closed circular HVS genome. As is characteristic of some other HVS-transformed nonproducer lymphoid cell lines, a large segment of unique light (L) DNA was missing in the persistent circular viral DNA present in 7710 cells. This deletion spanned at least 42.5 kilobases, corresponding to the segment between 12.3 and 50.7 map units of full-length, infectious virion L-DNA. The 7710 cells failed to induce tumors in athymic nude mice, but inbred rabbits inoculated with as few as 100 of these cells developed fatal lymphomas. Chromosomal analysis showed that tumors were due to the growth of donor cells. Cells recovered from one of the rabbits inoculated with 7710 cells also contained HVS DNA and, after in vitro culture, induced the same type of lymphoma when inoculated into two other III/J-strain rabbits. None of the previously described HVS-transformed cell lines have been able to induce tumors in either their host species or nude mice. Thus, our demonstration that the 7710 cell line is readily transplantable in syngeneic rabbits represents the first available model which allows analysis of many biological and molecular aspects of the in vivo oncogenicity of HVS.

Herpesvirus saimiri DNA in a lymphoid cell line established by in vitro transformation.

A lymphoid T-cell line (H1591) was established by infecting peripheral blood mononuclear cells from a cotton top marmoset with Herpesvirus saimiri OMI. Analysis of these in vitro-immortalized cells revealed nonintegrated, covalently closed circular viral DNA molecules in high multiplicities with substantial rearrangements and large deletions in their L-DNA (unique) regions. One subline, designated H1591 Er, contained circular viral DNA with one stretch of H-DNA (repetitive) and one of L-DNA; the L-DNA segment consisted of a linear fusion of a 53.2-kilobase-pair piece of L-DNA (left half of L-DNA) with a 15.2-kilobase-pair L-DNA fragment from the right end of the L-DNA region. The other subline, H1591 S, contained two short regions of L-DNA, each derived from the extreme ends of virion L-DNA. Both L-DNA regions of H1591 S cells contained inverted repetitions (15.0 +/- 0.2 and 9.1 +/- 4.7 kilobase pairs). The extensive deletions of L-DNA sequences in cell line H1591 indicate that at least 73% of the genetic information in H. saimiri is not required to maintain the persistence of viral DNA and the state of transformation in lymphoid T-cells.

Cloning of Herpesvirus saimiri DNA fragments representing the entire L-region of the genome.

Purified particles of Herpesvirus saimiri, a potent tumor-eliciting virus of primates, contain genomic DNA molecules (145-170 kb) consisting of a unique L-DNA region (112 kb) which is flanked by variable stretches of repetitive sequences (H-DNA). Restriction fragments representing the entire L-DNA of H. saimiri strain No. 11 were cloned in plasmid and bacteriophage vectors. The internal fragments of L-DNA generated by the enzymes EcoRI and KpnI were inserted into plasmid pACYC184, cosmid pJC81, or bacteriophage lambda derivative Charon 4A. The terminal parts of L-DNA, including the junctions between repetitive DNA and unique sequences, were cloned between the cleavage sites for KpnI and SmaI in the plasmid vector pWD7, which was constructed for this purpose. Molecular cloning allowed us to confirm and modify, in part, the existing cleavage maps of H. saimiri DNA. It provides a basis for future studies on virus replication and oncogenic transformation.

Expression of viral DNA in adenovirus type 12-transformed cells, in tumor cells, and in revertants.

The expression as cytoplasmic RNA of integrated human adenovirus type 12 (Ad12) DNA in transformed and tumor cell lines and in revertants was investigated. The transformed and tumor cells contained multiple copies of the viral genome, 3 to 22 copies per cell in different cell lines. The integrated Ad12 DNA molecules persisted intact or nearly intact and in most cases colinear with the virion DNA. In the revertant cell lines, which were derived from cell line T637 (22 copies of Ad12 DNA per cell), all of the Ad12 DNA molecules were lost (line F10) or only one copy and a fraction of a second copy persisted (line TR12). The size classes and map locations of Ad12-specific cytoplasmic RNAs in three Ad12-transformed hamster cell lines (T637, HA12/7, and A2497-3), in two revertant lines (F10 and TR12), in one Ad12-induced hamster (CLAC3), and in one rat brain tumor line (RBT12/3) were determined. Cytoplasmic RNA from uninfected B3 hamster cells and from human KB cells productively infected with Ad12 served as controls. In the latter control experiments, the RNA was isolated early or late postinfection. With respect to the amounts of Ad12-specific RNAs detected in cytoplasmic RNA from various Ad12-transformed or Ad12-induced tumor cell lines, we could not establish any correlations to the number of Ad12 genome copies integrated into the cellular DNAs. Thus, the expression of the integrated viral genomes in these lines was regulated by mechanisms more complicated than simple gene dosage effects. Using cloned fragments of Ad12 DNA as hybridization probes, we analyzed the cytoplasmic RNAs from the cell lines mentioned by electrophoresis on agarose gels, blotting, and DNA-RNA hybridization. For each transformed and tumor cell line, except for the revertants, several size classes of Ad12-specific cytoplasmic RNA were detected for the early E1, E2, and E4 regions of Ad12 DNA. Some of these size classes were similar but not identical to those observed in cytoplasmic RNA isolated early from human KB cells productively infected with Ad12. Only cell lines A2497-3, T637, and RBT12/3 contained several size classes of cytoplasmic RNA homologous to the E3 region of Ad12 DNA. Weak homologies to the E1 region of Ad12 DNA were also detected in the revertant lines F10 and TR12. Late regions of Ad12 DNA were expressed as cytoplasmic RNA in cell lines CLAC3 and RBT12/3. Weak homologies were detected between certain segments of the Ad12 genome (the EcoRI-B, -C, and -D fragments) and the cytoplasmic RNA from uninfected hamster cells. These homologies had no apparent counterpart at the level of DNA, perhaps because these homologies could be detected only due to an overrepresentation of RNA sequences. In preliminary experiments, we failed to detect the expression as cytoplasmic RNA of the so-called virus-associated RNA in transformed and tumor cell lines. Virus-associated RNA represents a population of low-molecular-weight RNAs that map at around 30 fractional length units on the viral genome.