Increases in neutral, Mg2+-dependent and acidic, Mg2+-independent sphingomyelinase activities precede commitment to apoptosis and are not a consequence of caspase 3-like activity in Molt-4 cells in response to thymidylate synthase inhibition by GW1843.

Thymidylate synthase (TS) inhibition causes cell death, and this enzyme is the target for the important chemotherapy regime 5-fluorouracil/leucovorin. GW1843 (1843U89) is a potent and specific folate analog TS inhibitor in clinical development. Because of the importance of TS as a chemotherapy target, we are studying the mechanism of TS inhibition-induced cell death by GW1843. Ceramide is a regulatory lipid generated by the action of sphingomyelinase and is believed to signal apoptosis. The role of the ceramide in apoptotic signaling was studied in Molt-4 human T-cell leukemia cells undergoing cell death after treatment with GW1843. In response to GW1843, Molt-4 cells undergo apoptosis with both acidic pH, Mg2+-independent sphingomyelinase (ASMase) and neutral pH, Mg2+-dependent sphingomyelinase (NSMase) activities elevated as early steps in the initiation of apoptosis before Molt-4 commitment to death. These activities lead to ceramide production with kinetics consistent with a role as an effector molecule signaling the initiation of apoptosis in Molt-4 cells. These changes were found to be independent of caspase 3-like (CPP32/apopain) activity and DNA degradation, but were not separable from membrane blebbing or cell lysis in this cell line. In this report, kinetic evidence is provided for a role of ceramide in initiating GW1843-induced cell death of Molt-4 T-cell leukemia cells.

DNA elements recognizing NF-Y and Sp1 regulate the human multidrug-resistance gene promoter.

Regulation of the human multidrug resistance gene (hMDR1) was studied by mapping DNA elements in the proximal promoter necessary for efficient transcription. Transient transfection analysis in tumor cell lines (HCT116, HepG2, and Saos2) of promoter deletions identified several regulatory domains. These cell lines expressed hMDR1 mRNA. Removal of an element between +25 and +158 reduced promoter activity by 2-3-fold, whereas deletion of sequences from approximately -5000 to -138 base pairs gave a approximately 2-fold increase. The activity of the hMDR1 promoter (-137 to +25) was comparable in activity to the SV40 early promoter and enhancer combination. Deletion of the hMDR1 promoter between -86 and -44 reduced activity by 5-10-fold, identifying an important regulatory region. This minimal region (-88 to -37) activated transcription when inserted upstream of a synthetic promoter, suggesting that it acts independently of other regulatory sequences. Two DNA elements within 85 base pairs of the transcriptional start site were required to confer efficient gene expression. A double-point mutation in the Y box (inverted CCAAT box) between -70 and -80 reduced activity of the promoter by 5-10-fold, and a single-point mutation at -52 within a GC-rich element reduced activity by 3-fold. Thus, both the Y-box and GC elements must each remain intact for optimal promoter activity. DNA-binding analyses suggest that the transcription factor NF-Y, but not YB-1 or c/EBP, is most likely responsible for controlling the activity of the Y-box element in these tumor cell lines. DNA-binding analyses also suggest that Sp1, alone or in combination with other nuclear factors, likely controls the activity of the GC element.

The anti-human immunodeficiency virus agent 3'-fluorothymidine induces DNA damage and apoptosis in human lymphoblastoid cells.

Patients infected with the human immunodeficiency virus experienced severe hematopoietic toxicity after treatment with the deoxynucleoside analog 3'-fluorothymidine (FLT). Using several methods for the analysis of genome integrity, including histochemical staining of the 3' ends of DNA and both conventional and pulsed-field agarose gel electrophoresis, we demonstrated that FLT caused extensive DNA fragmentation in CEM cells that was not observed when these cells were treated with other, less toxic thymidine analogs. In addition, a distinctive pattern of small DNA fragments that is characteristic of cells in the process of programmed cell death was observed in the genomic DNA of CEM cells treated with FLT. We conclude that FLT induces DNA fragmentation and apoptosis in a human cell line of hematopoietic origin, and we offer this observation as a possible explanation for the severe toxicity of FLT observed in vivo.

Association of nucleosome-free regions and basal transcription factors with in vivo-assembled chromatin templates active in vitro.

Using SV40 minichromosomes assembled in vivo, we have studied the relationship between a nucleosome-free promoter-region and initiation of transcription by RNA polymerase II on chromatin templates in vitro. Our data suggest that accessibility of DNA to transcription factors, programmed into the structure of the chromatin, is crucial for initiation of transcription. First, minichromosomes competent to be transcribed in vitro contained nucleosome-free promoter regions. Second, tsC219 minichromosomes, most of which contain the nucleosome-free promoter region, supported transcription more efficiently both in vivo and in vitro than wild-type minichromosomes, in which only a subset contain the nucleosome-free region. We have also identified basal transcription factors associated with the in vivo-assembled chromatin templates. A striking correlation was observed between minichromosomes associated with in vivo initiated RNA polymerases and those associated with the basal transcription factors TFIID and TFIIE/F, and to a lesser extent, TFIIB. Of these associated factors, only TFIID was poised for ready assembly into preinitiation complexes and therefore for subsequent initiation of transcription. However, an active chromatin template could also be maintained in the absence of the binding of TFIID. Finally, our data are consistent with the presence of TFIIF in elongating ternary complexes on the chromatin templates.

Activation of RNA polymerase II transcription by the specific DNA-binding protein LSF. Increased rate of binding of the basal promoter factor TFIIB.

While the components of the initiation complex at an RNA polymerase II basal promoter have been well characterized, few mechanistic studies have focused on how upstream DNA-binding, transcriptional activators influence protein assembly at the initiation site. Our analysis of basal transcription on both the simian virus 40 and adenovirus major late promoters demonstrates that two slow steps in initiation of transcription are the assembly of the general transcription factors TFIID and TFIIB onto the template DNA. On the simian virus 40 major late promoter, the rate of initiation complex formation is dramatically increased in the presence of the cellular transcriptional activator LSF. Direct analysis by band mobility shift assays demonstrates that LSF has no effect on the rate of binding, or the stability of TFIID on the promoter, predicting that LSF would not affect the template commitment step. Rather, kinetic analyses demonstrate that LSF reduces the lag in the rate of initiation complex formation attributable to the slow addition of TFIIB and suggest that LSF increases the rate of association of TFIIB with the committed template. In addition, LSF increases the total number of transcription complexes in long term assays, which is also consistent with LSF increasing the rate of association of TFIIB, where TFIIB is not saturating. These results indicate a mechanism for the activation of the initiation of RNA polymerase II transcription by one upstream activating protein, LSF. This mechanism may also be applicable to other activators that function in cases where limiting concentrations of TFIIB in the cell dictate slow binding of TFIIB.

In vitro initiation of transcription by RNA polymerase II on in vivo-assembled chromatin templates.

We have studied the initiation of transcription in vitro by RNA polymerase II on simian virus 40 (SV40) minichromosomal templates isolated from infected cells. The efficiency and pattern of transcription from the chromatin templates were compared with those from viral DNA templates by using two in vitro transcription systems, either HeLa whole-cell extract or basal transcription factors, RNA polymerase II, and one of two SV40 promoter-binding transcription factors, LSF and Sp1. Dramatic increases in numbers of transcripts upon addition of transcription extract and different patterns of usage of the multiple SV40 initiation sites upon addition of Sp1 versus LSF strongly suggested that transcripts were being initiated from the minichromosomal templates in vitro. That the majority of transcripts from the minichromosomes were due to initiation de novo was demonstrated by the efficient transcription observed in the presence of alpha-amanitin, which inhibited minichromosome-associated RNA polymerase II, and an alpha-amanitin-resistant RNA polymerase II, which initiated transcription in vitro. The pattern of transcription from the SV40 late and early promoters on the minichromosomal templates was similar to the in vivo pattern of transcription during the late stages of viral infection and was distinct from the pattern of transcription generated from viral DNA in vitro. In particular, the late promoter of the minichromosomal templates was transcribed with high efficiency, similar to viral DNA templates, while the early-early promoter of the minichromosomal templates was inhibited 10- to 15-fold. Finally, the number of minichromosomes competent to initiate transcription in vitro exceeded the amount actively being transcribed in vivo.

trans activation of the simian virus 40 late promoter by large T antigen requires binding sites for the cellular transcription factor TEF-1.

Simian virus 40 (SV40) T antigen stimulates the level of transcription from several RNA polymerase II promoters, including the SV40 late promoter. The mechanism of trans activation appears to be indirect since binding of T antigen to specific DNA sequences is not required. However, specific promoter elements that respond to T antigen have not previously been defined. We identified DNA sequences from the SV40 late promoter whose ability to stimulate transcription is induced by the expression of T antigen. In particular, the Sph I + II motifs of the SV40 enhancer can confer T-antigen inducibility to the normally uninducible herpes simplex virus thymidine kinase gene promoter when multiple copies of the sequence are inserted 5' of the transcription initiation site and TATA sequence. Binding sites for the cellular transcription factor TEF-1 and octamer binding proteins are contained within the Sph I + II motifs, as well as at other positions in the SV40 promoter. To study the role of individual protein-binding sites in trans activation by T antigen, mutations were constructed in various TEF-1 and octamer protein-binding sites of the SV40 late promoter. These mutations did not significantly affect basal promoter activity. However, mutation of all three TEF-1 sites prevented detectable activation by T antigen. DNase I footprinting of the mutated promoters with purified proteins demonstrated that inducibility by T antigen correlated with binding affinity of TEF-1 for the DNA and not with binding affinity of an octamer binding protein.

Transcription factor LSF binds two variant bipartite sites within the SV40 late promoter.

The HeLa transcription factor LSF has been purified by heparin-agarose and DNA affinity chromatography, and its DNA binding and transcription properties have been characterized. LSF is a 63-kD polypeptide that binds to two distinct bipartite sites within the SV40 promoter region. One binding site consists of GC motifs 2 and 3 within the 21-bp repeats (LSF-GC site), and the other consists of sequences centered 44 bp upstream of the major late initiation site, L325 (LSF-280 site). Four guanine residues within the LSF-GC site, when methylated, strongly interfere with LSF binding. Alteration of the spacing, but not the sequence, between the two directly repeated GC motifs dramatically reduces the binding affinity of LSF for the site. Thus, LSF appears to recognize directly repeated GC motifs, when their center-to-center distance is 10 bp. The LSF-GC and LSF-280 sites share limited sequence homology. Only half of the LSF-280 site contains a short GC-rich sequence homologous to the GC motif. However, the binding affinity of LSF to the two sites is similar. LSF activates transcription from the SV40 late promoter in vitro from initiation site L325, via its binding to the template DNA.