Regulation of human telomerase activity: repression by normal chromosome 3 abolishes nuclear telomerase reverse transcriptase transcripts but does not affect c-Myc activity.

Telomerase is required for the complete replication of chromosomal ends. In tumors, the human telomerase reverse transcriptase subunit (hTERT) is up-regulated, thereby removing a critical barrier for unlimited cell proliferation. To understand more about hTERT regulation, we measured hTERT RNA levels by quantitative reverse transcription (RT)-PCR. Telomerase-positive cell lines were found to contain between 0.2 and 6 molecules of spliced hTERT RNA per cell, whereas in telomerase-negative cells, the number of molecules was below the sensitivity of the assay (<0.004 molecules/cell). Intron-containing, immature hTERT RNA was observed only in nuclei of telomerase-positive cells, which suggests that hTERT RNA levels are transcriptionally regulated. Microcell transfer of a normal chromosome 3 into the human breast carcinoma cell line (21NT) abolishes telomerase activity and induces senescence. Endogenous hTERT transcripts were undetectable in the nuclei of 21NT-chromosome 3 hybrids, even in cells permanently expressing a transfected hTERT cDNA. However, chromosome 3 transfer did not affect the expression of green fluorescent protein reporter constructs driven by up to 7.4 kb of noncoding DNA flanking the 5' end of the hTERT gene. Because direct up-regulation of hTERT through c-Myc overexpression had previously been reported, we investigated whether chromosome 3 transfer affected c-Myc activity. An at least 30-fold reduction of immature intron-containing hTERT RNA was observed after the introduction of a normal chromosome 3, but expression levels of c-Myc, Mad1, and other c-Myc target genes were unchanged. Our results suggest that telomerase is regulated primarily at the level of hTERT transcription by complex mechanisms involving regulatory elements distant from the 5' flanking region, and that the putative hTERT repressor on chromosome 3 does not regulate the expression of hTERT through c-Myc or one of its coregulators.

Rearrangements of minisatellites in the human telomerase reverse transcriptase gene are not correlated with its expression in colon carcinomas.

Telomerase activation is crucial in human carcinogenesis. The limiting component of telomerase, the catalytic subunit (hTERT), is undetectable in normal somatic cells but present in most tumor cells, including the earliest stages of colon carcinoma. The mechanisms involved in the differential expression in normal and tumor cells are not understood. In normal cells hTERT expression is shut down by a repressor, and upregulation could be a consequence of cis-acting changes in the hTERT gene, making it resistant to repression. We have identified a polymorphic and a monomorphic minisatellite in the second intron of the hTERT gene, and polymorphic one in intron 6. The polymorphic minisatellite in intron 2 contains binding sites for c-Myc, which has been shown to upregulate hTERT transcription. Screening colon carcinoma DNAs for rearrangements of hTERT minisatellites we detected no changes in 33 samples from tumors, most of which express hTERT. This indicates that size rearrangements of the hTERT minisatellites are not required for telomerase expression in colon carcinomas. Minor changes and one LOH were seen in five tumors.

DNA binding specificity of different STAT proteins. Comparison of in vitro specificity with natural target sites.

STAT transcription factors are expressed in many cell types and bind to similar sequences. However, different STAT gene knock-outs show very distinct phenotypes. To determine whether differences between the binding specificities of STAT proteins account for these effects, we compared the sequences bound by STAT1, STAT5A, STAT5B, and STAT6. One sequence set was selected from random oligonucleotides by recombinant STAT1, STAT5A, or STAT6. For another set including many weak binding sites, we quantified the relative affinities to STAT1, STAT5A, STAT5B, and STAT6. We compared the results to the binding sites in natural STAT target genes identified by others. The experiments confirmed the similar specificity of different STAT proteins. Detailed analysis indicated that STAT5A specificity is more similar to that of STAT6 than that of STAT1, as expected from the evolutionary relationships. The preference of STAT6 for sites in which the half-palindromes (TTC) are separated by four nucleotides (N(4)) was confirmed, but analysis of weak binding sites showed that STAT6 binds fairly well to N(3) sites. As previously reported, STAT1 and STAT5 prefer N(3) sites; however, STAT5A, but not STAT1, weakly binds N(4) sites. None of the STATs bound to half-palindromes. There were no specificity differences between STAT5A and STAT5B.

Ectopic human telomerase catalytic subunit expression maintains telomere length but is not sufficient for CD8+ T lymphocyte immortalization.

Like most somatic human cells, T lymphocytes have a limited replicative life span. This phenomenon, called senescence, presents a serious barrier to clinical applications that require large numbers of Ag-specific T cells such as adoptive transfer therapy. Ectopic expression of hTERT, the human catalytic subunit of the enzyme telomerase, permits fibroblasts and endothelial cells to avoid senescence and to become immortal. In an attempt to immortalize normal human CD8(+) T lymphocytes, we infected bulk cultures or clones of these cells with a retrovirus transducing an hTERT cDNA clone. More than 90% of transduced cells expressed the transgene, and the cell populations contained high levels of telomerase activity. Measuring the content of total telomere repeats in individual cells (by flowFISH) we found that ectopic hTERT expression reversed the gradual loss of telomeric DNA observed in control populations during long term culture. Telomere length in transduced cells reached the levels observed in freshly isolated normal CD8(+) lymphocytes. Nevertheless, all hTERT-transduced populations stopped to divide at the same time as nontransduced or vector-transduced control cells. When kept in IL-2 the arrested cells remained alive. Our results indicate that hTERT may be required but is not sufficient to immortalize human T lymphocytes.

Elf-1 contributes to the function of the complex interleukin (IL)-2-responsive enhancer in the mouse IL-2 receptor alpha gene.

Lymphocytes regulate their responsiveness to IL-2 through the transcriptional control of the IL-2R alpha gene, which encodes a component of the high affinity IL-2 receptor. In the mouse IL-2R alpha gene this control is exerted via two regulatable elements, a promoter proximal region, and an IL-2-responsive enhancer (IL-2rE) 1.3 kb upstream. In vitro and in vivo functional analysis of the IL-2rE in the rodent thymic lymphoma-derived, CD4- CD8- cell line PC60 demonstrated that three separate elements, sites I, II, and III, were necessary for IL-2 responsiveness; these three sites demonstrate functional cooperation. Site III contains a consensus binding motif for members of the Ets family of transcription factors. Here we demonstrate that Elf-1, an Ets-like protein, binds to site III and participates in IL-2 responsiveness. In vitro site III forms a complex with a protein constitutively present in nuclear extracts from PC60 cells as well as from normal CD4- CD8- thymocytes. We have identified this molecule as Elf-1 according to a number of criteria. The complex possesses an identical electrophoretic mobility to that formed by recombinant Elf-1 protein and is super-shifted by anti-Elf-1 antibodies. Biotinylated IL-2rE probes precipitate Elf-1 from PC60 extracts provided site III is intact and both recombinant and PC60-derived proteins bind with the same relative affinities to different mutants of site III. In addition, by introducing mutations into the core of the site III Ets-like motif and comparing the corresponding effects on the in vitro binding of Elf-1 and the in vivo IL-2rE activity, we provide strong evidence that Elf-1 is directly involved in IL-2 responsiveness. The nature of the functional cooperativity observed between Elf-1 and the factors binding sites I and II remains unresolved; experiments presented here however suggest that this effect may not require direct interactions between the proteins binding these three elements.

Elf-1 and Stat5 bind to a critical element in a new enhancer of the human interleukin-2 receptor alpha gene.

The interleukin 2 receptor alpha-chain (IL-2R alpha) gene is a key regulator of lymphocyte proliferation. IL-2R alpha is rapidly and potently induced in T cells in response to mitogenic stimuli. Interleukin 2 (IL-2) stimulates IL-2R alpha. transcription, thereby amplifying expression of its own high-affinity receptor. IL-2R alpha transcription is at least in part controlled by two positive regulatory regions, PRRI and PRRII. PRRI is an inducible proximal enhancer, located between nucleotides -276 and -244, which contains NF-kappaB and SRE/CArG motifs. PRRII is a T-cell-specific enhancer, located between nucleotides -137 and -64, which binds the T-cell-specific Ets protein Elf-1 and HMG-I(Y) proteins. However, none of these proximal regions account for the induction of IL-2R alpha transcription by IL-2. To find new regulatory regions of the IL-2R alpha gene, 8.5 kb of the 5' end noncoding sequence of the IL-2R alpha gene have been sequenced. We identified an 86-nucleotide fragment that is 90% identical to the recently characterized murine IL-2-responsive element (mIL-2rE). This putative human IL-2rE, designated PRRIII, confers IL-2 responsiveness on a heterologous promoter. PRRIII contains a Stat protein binding site that overlaps with an EBS motif (GASd/EBSd). These are essential for IL-2 inducibility of PRRIII/CAT reporter constructs. IL-2 induced the binding of Stat5a and b proteins to the human GASd element. To confirm the physiological relevance of these findings, we carried out in vivo footprinting experiments which showed that stimulation of IL-2R alpha expression correlated with occupancy of the GASd element. Our data demonstrate a major role of the GASd/EBSd element in IL-2R alpha regulation and suggest that the T-cell-specific Elf-1 factor can serve as a transcriptional repressor.

Mouse interleukin-2 receptor alpha gene expression. Interleukin-1 and interleukin-2 control transcription via distinct cis-acting elements.

We have shown that interleukin-1 (IL-1) and IL-2 control IL-2 receptor alpha (IL-2R alpha) gene transcription in CD4-CD8- murine T lymphocyte precursors. Here we map the cis-acting elements that mediate interleukin responsiveness of the mouse IL-2R alpha gene using a thymic lymphoma-derived hybridoma (PC60). The transcriptional response of the IL-2R alpha gene to stimulation by IL-1 + IL-2 is biphasic. IL-1 induces a rapid, protein synthesis-independent appearance of IL-2R alpha mRNA that is blocked by inhibitors of NF-kappa B activation. It also primes cells to become IL-2 responsive and thereby prepares the second phase, in which IL-2 induces a 100-fold further increase in IL-2R alpha transcripts. Transient transfection experiments show that several elements in the promoter-proximal region of the IL-2R alpha gene contribute to IL-1 responsiveness, most importantly an NF-kappa B site conserved in the human and mouse gene. IL-2 responsiveness, on the other hand, depends on a 78-nucleotide segment 1.3 kilobases upstream of the major transcription start site. This segment functions as an IL-2-inducible enhancer and lies within a region that becomes DNase I hypersensitive in normal T cells in which IL-2R alpha expression has been induced. IL-2 responsiveness requires three distinct elements within the enhancer. Two of these are potential binding sites for STAT proteins.

Characterization of a new tissue-specific transcription factor binding to the simian virus 40 enhancer TC-II (NF-kappa B) element.

We have biochemically and functionally characterized a new transcription factor, NP-TCII, which is present in nuclei from unstimulated T and B lymphocytes but is not found in nonhematopoietic cells. This factor has a DNA-binding specificity similar to that of NF-kappa B but is unrelated to this or other Rel proteins by functional and biochemical criteria. It can also be distinguished from other previously described lymphocyte-specific DNA-binding proteins.

Minimal growth requirements of mature T lymphocytes: interleukin (IL)-1 and IL-6 increase growth rate but not plating efficiency of CD4 cells stimulated with anti-CD3 and IL-2.

We show that interleukin (IL)-2 is necessary and sufficient for the proliferation of both CD4 and CD8 subsets of peripheral murine T cells activated by plastic-bound anti-CD3 monoclonal antibodies (mAb). The frequency of proliferating cells (f) was 0.32 for CD4 cells and 0.63 for CD8 cells. These frequencies were not increased by the addition of IL-1 or IL-6, alone or in combination. These cytokines were unable to induce responsiveness to IL-2 in T cells confirming that they cannot substitute for the signal delivered via the TcR/CD3 complex. On the other hand, IL-1 and IL-6 increase the growth rate of CD4 cells. The addition of IL-6 significantly lowered the mean doubling time (dt) of CD4 cells (dt: 26 h vs. 38 h in the presence of IL-2 alone, p less than 0.01), while the addition of IL-1, ineffective by itself, combined with IL-6 further increased the growth rate of CD4 cells (dt: 23 h, p less than 0.001). The growth rate of CD8 cells stimulated with anti-CD3 and IL-2, was markedly faster than that of CD4 cells (dt: 18 h vs. 38 h, p less than 0.001) and was not significantly influenced by addition of IL-1 and/or IL-6.

Control of IL-2 receptor-alpha expression by IL-1, tumor necrosis factor, and IL-2. Complex regulation via elements in the 5' flanking region.

We have analyzed the mechanisms by which IL-1, IL-2, and TNF regulate expression of IL-2R alpha chain in a rodent T cell line. All three cytokines induce detectable IL-2R alpha mRNA by themselves, but there is strong synergy between IL-1 or TNF, on the one hand, and IL-2, on the other. The earliest phase of induction by IL-1 is independent of protein synthesis. IL-1, but not TNF, also stimulates transient secretion of IL-2. This leads to an autocrine stimulation of a further increase in IL-2R alpha mRNA levels. When IL-2 secretion has dropped off, continued IL-2R alpha expression requires both IL-2 and IL-1. Most or all of this regulation is due to changes in the rate of transcription of the IL-2R alpha gene. The response to IL-1 and IL-2 depends on a segment in the IL-2R alpha 5' flanking region, upstream of all cis-acting regulatory elements previously identified in the human gene.

Activity and interleukin 1 responsiveness of SV40 enhancer motifs in a rodent immature T cell line.

We have analysed the enhancer activity and the interleukin 1 (IL1) responsiveness of individual motifs of the SV40 enhancer in an immature rodent T cell line, PC60. Transient transfection assays showed that tetramers of GT-I plus GT-IIC motifs, the TC-II or the P motif have significant enhancer activity in PC60, while neither Octamer nor SphI+II motifs have a detectable effect on promoter strength. Two motifs, TC-II and P, strongly respond to stimulation by IL1. DNase I and methylation protection experiments with nuclear extracts show specific footprints in the TC-II region of the SV40 enhancer. Exposure of PC60 cells to IL1 increases their intensity. The TC-II sequence forms several complexes detected in band shift assays. The molecules involved all have similar sequence specificity as NF-kappa B. Surprisingly, band shifts with extracts from control or IL1 treated cells differ only slightly. However, if GTP is added to the binding reactions the intensity of bands formed by extracts from control cells is strongly reduced, whereas extracts from IL1 treated cells form a single retarded complex that co-migrates with NF-kappa B from a pre-B cell line. The results suggest that in PC60 IL1 induces NF-kappa B activity by activating molecules that are already in the nucleus.

Loss of interleukin 2 dependence in cloned interleukin 2-dependent rat T lymphocyte x BW5147 hybridomas is not associated with segregation of a specific pair of rat chromosomes.

A fusion between the mouse AKR thymoma BW5147 and a culture of homogeneously OX8+ (CD8) rat T lymphoblasts yield interleukin (IL) 2-dependent T cell hybridomas when selected in HAT medium supplemented with IL 2-containing supernatants of concanvalin A-activated cells and dexamethasone. IL 2-independent variants can be selected from cloned IL2-dependent hybrids in the absence of conditioned medium. Karyotype analysis was used to test a previously proposed hypothesis according to which IL2-independent variants arise through loss of a specific cytotoxic T lymphocyte (rat) chromosome carrying a gene responsible for IL2 dependence. Comparison of karyotypes of several independently derived hybrids with those of their IL 2-independent variants showed that the hybrids contain at least one homologue of all rat chromosomes, and that no pair of rat chromosomes is consistently absent in the IL 2-independent variants.

Interleukin 1 and tumor necrosis factor enhance transcription from the SV40 early promoter in a T cell line.

Interleukin 1 (IL 1) and tumor necrosis factor (TNF) increase the expression of a number of T lymphocyte-specific genes in the T-cell hybrid PC60. We show here that human IL 1 alpha and 1 beta, mouse IL 1 alpha and mouse TNF-alpha strongly enhance expression of reporter genes transcribed from the SV40 early promoter in this cell line. We found that IL 1 and TNF each induce up to a 30-fold increase in the rate of transcription, resulting in a proportional increase of mRNA and protein levels. Induction with IL 1 and TNF is detectable after 1 h and reaches a plateau after about 15 h. Removal of these factors from the culture medium results in complete reversion. Induction with IL 1, but not with TNF, can be inhibited with an inhibitor of IL 1 binding. The effects of both factors are not impaired by the protein synthesis inhibitor cycloheximide, suggesting that they stimulate expression from the SV40 early promoter by activation of preexisting transcription factors.

Recombinant tumor necrosis factor can induce interleukin 2 receptor expression and cytolytic activity in a rat x mouse T cell hybrid.

Tumor necrosis factor (TNF), an endotoxin-induced macrophage monokine, is known for its cytotoxic and cytostatic effect on some tumor cell lines. Here we show that highly purified recombinant TNF, in combination with interleukin 2 (IL2), can induce IL2 receptor expression and cytolytic activity in a rat x mouse T cell hybrid (PC60). Previously, it was shown that IL1 had a similar effect on PC60 cells. The ability of TNF to co-induce IL2 receptor expression suggests that it may play a role in the activation of certain lymphoid effector cells. This observation augments the growing list of biological activities attributed to TNF.

Granzyme A secretion by normal activated Lyt-2+ and L3T4+ T cells in response to antigenic stimulation.

Granzyme A is a serine esterase initially isolated from cytoplasmic granules of cytolytic T cell lines (Masson, D. et al., EMBO J. 1986. 5: 1595). Among normal T lymphocytes activated by allogeneic stimulation it is found in both Lyt-2+ and L3T4+ subsets in comparable amounts. Here we show that normal alloantigen-activated T cells secrete the enzyme specifically when mixed with appropriate stimulator cells. Specific enzyme release is dependent on external calcium and removal of calcium blocks further secretion within a few minutes. Both Lyt-2+ and L3T4+ cells specifically secrete the enzyme with similar rates, and anti-L3T4 and anti-Lyt-2 antibodies block secretion by the responder cells expressing these markers. Anti-LFA-1 antibodies, on the other hand, block secretion by either subset.

Perforin is present only in normal activated Lyt2+ T lymphocytes and not in L3T4+ cells, but the serine protease granzyme A is made by both subsets.

We show that among the subsets of peripheral T lymphocytes (Lyt2+ L3T4- and L3T4+ Lyt2- cells) activated in short-term cultures by stimulation with H-2 incompatible leukocytes 97% of the cytolytic activity and all detectable perforin activity resides in the Lyt2+ cells. But both populations contain approximately equal amounts of a serine protease, granzyme A, the expression of which was previously thought to be restricted to cytolytic T lymphocytes.

Dissociation of interleukin 2-dependent and -independent B cell proliferation with monoclonal anti-interleukin 2 receptor antibody.

In a recent report from our laboratory (J. Exp. Med. 1984. 160: 1070), it has been shown that B cells which have been activated with lipopolysaccharide (LPS) plus anti-Ig antibodies express interleukin 2 (IL2) receptors and proliferate in response to pure IL2. In the present study, we have investigated the role of IL2 during proliferative B cell responses supported by various T cell supernatants (SN) as well as by LPS itself. The following results were obtained: (a) B cells activated with LPS plus anti-Ig were found to proliferate in response to either recombinant IL2, T cell SN or fresh LPS; (b) a monoclonal anti-IL2 receptor antibody (PC61) could completely inhibit the effects of recombinant IL2 or various T cell SN (cloned T helper cell SN, EL4 SN, concanavalin A-spleen cell SN or mixed leukocyte culture SN), but did not interfere with the effect of LPS; (c) B cells activated with LPS or LPS plus anti-Ig were not found to secrete detectable amounts of IL2 by themselves. Thus, the data demonstrate that LPS-stimulated B cell proliferation requires neither B cell autocrine nor T cell-derived IL2. On the other hand, with regard to LPS plus anti-Ig-activated B cells, IL2 appeared to be the only growth-promoting activity that could be detected in the supernatants obtained from total normal spleen cell populations.