Immature thymocytes undergoing receptor rearrangements are resistant to an Atm-dependent death pathway activated in mature T cells by double-stranded DNA breaks.

Immature CD4(+)CD8(+) thymocytes rearrange their T cell receptor (TCR)-alpha gene locus to generate clonotypic alpha/beta TCR, after which a few cells expressing selectable TCR are signaled to further differentiate into mature T cells. Because of requirements for self-tolerance, immature CD4(+)CD8(+) thymocytes are programmed to die in the thymus in response to a variety of stimuli that do not induce death of mature T cells. We now demonstrate that, in contrast to all previously described stimuli, immature CD4(+)CD8(+) thymocytes are selectively more resistant than mature T cells to apoptotic death induced by DNA intercalating agents. Importantly, we demonstrate that DNA intercalating agents induce double-stranded DNA breaks in both immature thymocytes and mature T cells, but immature thymocytes tolerate these DNA breaks, whereas mature T cells are signaled to die by an Atm-dependent but p53-independent death mechanism. Thus, our results indicate that absence of an Atm-dependent but p53-independent pathway allows immature thymocytes to survive double-stranded DNA breaks. It is likely that the unique ability of immature thymocytes to survive DNA-damaging intercalating agents reflects their tolerance of double-stranded DNA breaks that occur normally during antigen receptor gene rearrangements.

Measurement of low-abundance cytokine mRNA in cells of murine lymphoid organs: a new quantitative reverse transcription/polymerase chain reaction method.

To investigate cytokine regulation in cells of freshly excised lymphoid tissues, rigorous quantitative reverse transcription/polymerase chain reaction (QRT-PCR) assays were developed to measure attomole (10(-18) mol) amounts of the mRNA for seven cytokines: interleukin-1alpha (IL-1alpha), IL-1beta, tumor necrosis factor alpha (TNFalpha), interferon gamma (IFNgamma), transforming growth factor beta (TGFbeta), IL-2 and IL-6. RNA was purified from single-cell suspensions of immune tissues (spleen, thymus and resident peritoneal cells). Data are presented demonstrating the utility of these assays for quantifying basal levels of all seven cytokine mRNAs in the freshly isolated splenocytes and thymocytes. Studies to establish the usefulness of these assays for measuring changes in the levels of cytokine mRNA focused on IL-1alpha, IL-1beta, TNFalpha and IL-2 in splenocytes, thymocytes and resident peritoneal cells. Using the QRT-PCR assays developed, levels of cytokine mRNA could be quantified in RNA samples obtained both from freshly isolated cells and from cells following short-term (

Recent advances in the study of rTS proteins. rTS expression during growth and in response to thymidylate synthase inhibitors in human tumor cells.

The rTS proteins have now been shown to be expressed in a variety of cell lines, with expression of rTS beta being found elevated in three cell lines which are resistant to TS inhibitors (3, 4) (Figure 1). In one of these cell lines (K562 B1A), the cells were selected for resistance to MTX, which has a primary site of action on DHFR, but was found to be cross-resistant to FUdR (4). The other two cell lines were selected for resistance to either 5-fluorouracil (H630-1) or a combination of ZD1694 and FU. In each case, elevation of rTS beta appears to be a selected response to thymidylate stress. In HCT-8 and HCT-8/DF2 cells, treatment of cells for a short period of time (2 hr) resulted in the elevation of rTS beta levels, again suggestive that expression of rTS beta is a response to thymidylate stress. rTS beta appears to be regulated with cell growth, its levels increasing at mid-log and at late-log/saturation phase in H630 and H630-1 cells (Fig. 2), and increasing with late-log in several other cell lines as well (Fig. 3). The increase in rTS beta is suggestive of a cellular function associated with a state where growth is no longer desirable, reminiscent of the starvation-sensing protein homolog RSPA in E. coli (22). While this relationship would not explain the spike in rTS beta levels in mid-log H630 and H630-1 cells, it does make sense if the rTS proteins (particularly rTS beta) are involved in down-regulating thymidylate biosynthesis. The potential mechanism of this down-regulation may be speculated to be the catabolism of some precursor for thymidylate biosynthesis or some direct effect upon TS through modulation by some other ligand, either a metabolite or another protein. Studies on the expression of rTS proteins in clinical specimens indicate that rTS beta is expressed at high levels in kidney and kidney tumor (Dolnick, unpublished results). Given the physiologic role of the kidney, high level expression of rTS in this organ is consistent with a role in a catabolic pathway. Since down-regulation of TS activity is expected to increase sensitivity to TS inhibitors, a role for rTS beta in directly down-regulating TS activity in the biochemical sense would seem unlikely. However, the manner of biochemical TS down-regulation may make a difference. In the TS- Cl/Cl cell line, there are two mutations in TS which likely reduce affinity for N-5,10-methylene tetrahydrofolates (23). This cell line is highly resistant to MTX, yet is still tumorigenic in vivo (24), and supplying the cells with high levels of exogenous folate can restore TS function (23). Thus in TS- Cl/Cl cells, the TS phenotype is conditionally dependent upon the presence of high levels of exogenous folate. This suggests that a role of rTS proteins as conditional down-regulators of TS, perhaps through modulating folate binding, may be possible. Two cell lines (K562 B1A and H630-1) that overproduce rTS beta have altered sensitivity to TS inhibitors that differ depending upon the nature of the inhibitor. The K562 B1A cell line was found to be approximately 2000-fold resistant to ZD1694 and BW1843U89 (120 hr exposures), but only three-fold resistant to AG331. The H630-1 cell line is approximately 30-fold resistant to BW1843U89 (120 hr exposure) and 40-fold resistant to ZD1694 (120 hr exposure), but only eight-fold resistant to AG331. Since K562 B1A cells overproduce rTS beta (2), but have no significant alterations in FPGS activity, the possibility that rTS may affect folate binding remains a hypothesis worth examining. The recent discovery that TS is a phosphoprotein and that it is nuclear as well as cytoplasmic (21) raises the possibility that the phosphorylation state of TS may regulate one of its cellular functions, and that the subcellular localization of this enzyme is regulated as well. Since rTS proteins have HSP with proteins that participate in kinase/phosphatase reactions, this also seems to be an avenue worthy of future investigation. (ABSTRACT TRUNCATED

Naturally occurring antisense RNA.

Within the last few years a number of mammalian genes have been found for which there exist naturally occurring "antisense" RNA species with complementarity to mRNAs. Effects of antisense RNA on "sense" RNA have yet to be established. Nevertheless, it is apparent that mammalian cells have devoted genetic information and machinery to processing RNA:RNA hybrids, and it is becoming clear that there may be many more genes than previously suspected to which natural antisense RNAs exist. If naturally occurring antisense RNAs are mediators of alterations in gene expression, the question arises as to whether these pathways can be exploited pharmacologically.

Alternate splicing of the rTS gene product and its overexpression in a 5-fluorouracil-resistant cell line.

rTS is a gene with convergent and overlapping transcription relative to thymidylate synthase (TS). Screening of a cDNA library has identified a second rTS RNA (rTSbeta) which is related to a previously described rTS RNA (rTSalpha). rTSbeta differs from rTSalpha by the insertion of an extra 116 nucleotides after base 128 of rTSalpha and an altered 3'-noncoding region such that rTSbeta RNA is not complementary to TS mRNA. rTSalpha and rTSbeta RNAs have open reading frames predicted to encode proteins of 361 and 416 amino acids, respectively. Antibody to rTSbeta protein detects two proteins of the predicted molecular weight in the H630 colon tumor cell line. rTSbeta is overexpressed 40-70-fold in a 5-fluorouracil-resistant H630 subline compared with 7-8-fold for TS and rTSalpha.

Expression of rTS correlates with altered growth regulation of thymidylate synthase.

The recently discovered rTS gene is convergent with and overlaps the thymidylate synthase gene in the antisense orientation. K562 B1A and KB 1BT are two methotrexate-resistant cell lines that have amplified genes for dihydrofolate reductase. K562 B1A cells have increased levels of rTS mRNA and protein compared with their parental K562 cells, whereas KB 1BT cells show unaltered rTS expression compared with their parental (KB) cells. Altered levels and loss of growth-regulation of in situ thymidylate synthase activity were found in methotrexate-resistant K562 B1A cells but not in K562, KB, and KB 1BT cells. These data point to a link between rTS and the regulation of TS activity.

rTS gene expression is associated with altered cell sensitivity to thymidylate synthase inhibitors.

rTS is a recently discovered gene, phylogenetically conserved and found to be expressed in a wide variety of cell lines. rTS has also been found to be overexpressed in two cell lines resistant to FU and to MTX. The MTX-resistant cell line was found to have a high degree of cross resistance to several TS inhibitors. An apparent paradox to this correlation of rTS overexpression and resistance to TS inhibitors is the observation that expression of transfected rTS alpha results in enhanced sensitivity of cells to the TS inhibitor prodrug TFT and a modest increase in resistance to FUdR. Since immunoprecipitation of TS leads to the co-immunoprecipitation of two proteins within the expected molecular weight range of the two rTS proteins, it may be that both proteins bind to TS in vivo and modify its activity. Preliminary data substantiate this conclusion. It is conceivable that the ratio of the two rTS proteins associated with TS in vivo may differentially alter TS activity depending upon their stoichiometry or possibly posttranslational modification. Thus it may be possible for rTS to confer greater sensitivity to one pyrimidine analog (e.g., TFT) which is a product analog but to increase resistance or have a minor effect on a substrate analog (e.g., FdUMP) by stabilizing different conformations of TS. The structure of the rTS proteins suggests they are expected to have catalytic activity which involves proton abstraction from an alpha-carbon of a carboxyl group. Whether this enzyme activity is functional and related to pyrimidine metabolism awaits further study.

Subclonal heterogeneity of the multidrug resistance phenotype in a cell line expressing antisense MDR1 RNA.

A multidrug resistant (MDR) cell line was transfected with an antisense MDR1 expression vector and transfectant clones were analyzed for reversion of the MDR phenotype. Only one of 10 antisense-expressing transfectants showed a reduction in drug resistance, MDR1 mRNA and P-glycoprotein. Observations made using rhodamine-123, a fluorescent substrate for P-glycoprotein, revealed that dye retention in individual cells was highly variable within this antisense-expressing clone. Subpopulations were established from the original clone based on differences in rhodamine-123 retention. Rhodamine-123 retention varied inversely with levels of P-glycoprotein and MDR1 mRNA. All subpopulations expressed similar levels of antisense MDR1 RNA yet had dramatic differences in MDR1 mRNA levels. Analysis of vector integration site restriction fragment length polymorphisms confirmed that all populations originated from the same transfectant clone. Nuclear run-on analysis indicated that the mdr1 gene is transcribed at the same rate in all populations, suggesting that the reduction in MDR1 mRNA is mediated posttranscriptionally. Cells with the greatest reduction in MDR1 mRNA accumulate distinct antisense RNA transcripts in the nuclear RNA fraction, suggesting that antisense effectiveness in this system is associated with a nuclear event or process. These results reveal that antisense RNA activity is not necessarily distributed equally within a clonal population.

Regulation of folate-binding protein gene expression by DNA methylation in methotrexate-resistant KB cells.

Folate-binding protein (FBP) is responsible for the cellular transport of folate and methotrexate (MTX) in human KB (nasopharyngeal epidermoid carcinoma) cells. The levels of membrane-associated FBP and FBP mRNA are decreased 70-80% in an MTX-resistant KB subline (KB1BT) (Hsueh C-T and Dolnick BJ, Oncol Res 4: 497-505, 1992). Southern blot analysis did not reveal any differences in FBP gene organization or copy number between KB1BT and KB cells. However, there was a 70% decrease in the FBP gene transcription rate and no change in FBP mRNA stability in KB1BT cells. Assessing genomic DNA methylation by MspI and HpaII restriction analysis suggested that the FBP gene in KB1BT cells was more methylated than in KB cells. These alterations in the expression, transcription rate and DNA methylation state of the FBP gene did not change when KB1BT cells were grown in the absence of MTX for 8 months (MTX-free KB1BT). When MTX-free KB1BT cells were exposed to 2.5 microM 5-aza-2'-deoxycytidine for 72 hr, the FBP gene became hypomethylated and the levels of membrane-associated FBP and FBP mRNA increased by 2- to 3-fold. These data indicate that decreased FBP gene expression in KB1BT cells results from increased DNA methylation.

Quantitative evaluation of intracellular sense: antisense RNA hybrid duplexes.

Previous studies have demonstrated that for an antisense RNA to be effective in attenuating gene expression, a large but indeterminate excess of antisense RNA is required. To quantitatively evaluate RNA hybrid duplex formation, expression vectors containing antisense dihydrofolate reductase (DHFR) cDNAs were transfected into KB and KB-1BT (a DHFR overexpressing variant) cells and transfectants expressing antisense transcripts of exon 1 through intron I (ex1-I) or exons 1 through 4 (ex1-4) were analyzed for hybrid duplex formation. Stable duplexes were detectable in KB-1BT but not in KB cells. Approximately 5-9% of antisense ex1-I RNA and 20-37% of antisense ex1-4 RNA were found in duplexes. The amount of each hybrid duplex RNA was found to be a linear function of intracellular single-stranded antisense RNA levels and a hybrid index, Hs:as, was devised to describe this relationship. Based upon the value of Hs:as for each antisense RNA:mRNA duplex, it is calculated that an approximate 2,800- and 600-fold excess of ex1-I and ex1-4 antisense RNA are respectively required for 50% of DHFR mRNA to be present in duplexes. Results support the hypothesis that intracellular sense:antisense RNA hybrid duplex formation is inefficient and dependent upon the levels, lengths and possibly the structures of the RNAs involved.

5-Fluorouracil alters dihydrofolate reductase pre-mRNA splicing as determined by quantitative polymerase chain reaction.

A quantitative polymerase chain reaction (PCR) assay has been developed to determine the absolute and relative amounts of each dihydrofolate reductase RNA species present at different stages of splicing (i.e., pre-mRNA, splicing intermediate, and mRNA). The ratios of each RNA species as measured by quantitative PCR have been confirmed by S1 nuclease mapping analysis. Quantitative PCR studies reveal a concentration-dependent decrease in the levels of dihydrofolate reductase mRNA and a splicing intermediate but little change in pre-mRNA levels after long term exposure of cells to 5-fluorouracil (FUra). The observed changes correlate with the extent of FUra incorporation into RNA and with cytotoxicity. These results, together with previous data from our laboratory, provide the first direct evidence that FUra incorporation into RNA can cause inhibition of pre-mRNA splicing in vivo. Inhibition of pre-mRNA splicing is thus a likely additional mechanism by which FUra incorporation into RNA may lead to growth inhibition and cell death.

Altered folate-binding protein mRNA stability in KB cells grown in folate-deficient medium.

Folate-binding protein (FBP), a high-affinity folate receptor, is responsible for cellular accumulation of folate and folate analogs such as methotrexate in human KB (nasopharyngeal carcinoma) cells. Both FBP and FBP mRNA increase 3- to 5-fold when KB cells are grown in folate-deficient (less than 10 nM folate) medium (KB-FD), compared with growth in standard folate-replete medium containing at least 2 microM folate (KB-FR). The possible mechanisms of enhanced FBP gene expression in KB-FD were examined in this study. Southern blot analysis revealed no significant change in the FBP gene organization or copy number in the KB-FD DNA. While hypomethylation of the FBP gene was observed in KB-FD DNA, relative to KB-FR DNA, exposure of KB-FR to the DNA methylation inhibitors did not result in elevated FBP mRNA levels. The transcriptional rate of the FBP gene was the same in KB-FR and KB-FD. RNA half-life studies indicated that the half-life of FBP mRNA in KB-FD was increased approximately 2.5-fold, compared with KB-FR. Thus, the increase in the steady-state levels of FBP mRNA in KB-FD can be attributed partly to increased FBP mRNA stability.

Cloning and characterization of a naturally occurring antisense RNA to human thymidylate synthase mRNA.

Based upon reverse transcription and polymerase chain reaction results with human KB cell RNA, a cDNA (i.e., 3'rTS1, 1557 nt) with complementarity to thymidylate synthase mRNA was cloned and sequenced. Northern blot analysis showed that 3'rTS1 corresponded to a cytoplasmic 1.8 kb RNA found in several tumor cell lines. The remaining 5'region of this antisense RNA was cloned by a RACE (Rapid Amplification of cDNA Ends) procedure. A full length cDNA (i.e., rTS, 1811 nt) was generated by splicing 3'rTS1 with RACE-generated cDNA. rTS RNA is likely a mRNA that contains four open reading frames. Based upon sequence analysis of the RACE cDNAs and the rTS cDNA, rTS RNA is likely processed from a gene containing at least six introns. Northern blot analysis indicates rTS RNA is expressed in a variety of human tumor cell lines and an aberrant from is expressed in a methotrexate-cell line.

Effects of 5-fluorouracil on mRNA.

Currently, there are a number of studies which suggest that FU can have pronounced effects on mRNA and its metabolism. However, the relevance of these changes to the antitumor effect of FU are still not clear. Generally, the mRNAs which have been studied to date involve those genes which are associated with the TS-directed effects of FU and have generally been limited to the changes in mRNA levels. The recent development of PCR methodology to investigate changes in pre-mRNA and splicing provides the tool to study a number of RNA effects of FU simultaneously. The major question is which mRNAs are important for study. DHFR mRNA has a half life of 11.5 in KB1BT cells (Will and Dolnick, 1989) and is thus, on a kinetic basis alone, unlikely to provide a significant RNA target for RNA-directed effects of FU. There is a greater likelihood that shorter lived mRNAs which not only turnover rapidly, but are important to cell proliferation will eventually be shown to be key targets for the effects of FU at the RNA level. Interestingly, many of the growth factors are encoded by short-lived and tightly regulated mRNAs (e.g. GM-CSF, Shaw and Kamen, 1986). In fact the half-lives of some of these mRNAs are regulated by U-rich sequences in their 3'-noncoding regions. The presence of U-rich sequences in these growth factor mRNAs and the small nuclear RNAs suggests these are worthwhile targets for studies, which could now be performed on clinical samples. Laboratory data which shows alterations in the small nuclear RNAs, under conditions which only provide for very low-level substitution of U residues by FU also suggest that RNA effects of FU may be a much more tightly related to cytotoxicity in vivo than previously thought.

Enhanced sensitivity to G418 of human KB cells adapted to certain media and sera.

Sensitivity to the neomycin derivative G418 was determined for a human cell line, KB 3-1, that had been adapted to six different combinations of media and sera. The results indicate that while the plating efficiency is similar for all conditions, the susceptibility to G418 can differ markedly depending on the particular combination of media and sera used. This suggests that in experiments using neomycin resistance as a selectable marker, conditions may be found where the amount of G418 required for selection and maintenance of transfected cell lines can be reduced, providing a significant savings.

Mutation(s) of the thymidylate synthase gene of human adenocarcinoma cells causes a thymidylate synthase-negative phenotype that can be attenuated by exogenous folates.

Biological characterization of a human colon adenocarcinoma cell line deficient in thymidylate synthase (TS-) is described. The clone, designated TS-C1/C1, was derived from the parental line GC3/C1 by selection in medium containing aminopterin, thymidine (dThd), and low concentrations of 5-formyltetrahydrofolate (5-CHO-H4PteGlu), and was subsequently reselected by single-step cloning in 500 microM methotrexate in the presence of dThd. This clone retained its TS- phenotype, was highly resistant to methotrexate (greater than 100,000-fold), and remained tumorigenic in mice (P.J. Houghton, et al., Proc. Natl. Acad. Sci. USA, 86: 1377-1381, 1989). In studies reported, it is shown that high levels of exogenous folate can support the growth of the TS- C1/C1 clone in the absence of dThd. Activation of dTMP biosynthesis de novo was demonstrated within 6 h of exposing cells to 20 microM [6R,S]5-CHO-H4PteGlu, and greater than or equal to 80% of activity was lost within 24 h of removing this folate from the medium. The labeling index was determined by autoradiographic techniques using [6-3H]2'-deoxyuridine. None of the greater than 6,000 cells radiolabeled in the absence of [6R,S]5-CHO-H4PteGlu, whereas 33.5% labeled in the presence of 20 microM exogenous folate. Relative to the parental (TS+) clone, there was a greater than 87,500-, an 8,182-, and a 425-fold higher requirement for 5-methyltetrahydrofolate ([6R,S]5-CH3-H4PteGlu), PteGlu, and [6R,S]5-CH3-H4PteGlu to support 50% maximal colony formation in the absence of dThd. Quantitative analysis of the combined pools of 5,10-methylenetetrahydrofolate (CH2-H4PteGlun) and H4PteGlun showed that parental GC3/C1 cells had higher endogenous folate pools compared to TS-C1/C1 cells [168 +/- 40 (SD) and 10.9 +/- 0.3 fmol/10(6) cells, respectively]. Qualitatively the distribution of polyglutamate species and their redistribution in cells exposed to 20 microM [6R,S]5-CHO-H4PteGlu were similar in the two lines. Analysis of pools in a second, independently derived, TS- clone (TS-C3/C3, a transcription-negative mutant) demonstrated undetectable levels of CH2-H4PteGlun and H4PteGlun. This line cannot be rescued by exogenous folate. The data thus suggest that deletion of dTMP synthase activity may cause redistribution of reduced folate pools. In cytosolic extracts from parental GC3/C1 (TS+) cells, [6R]CH2-H4PteGlu1 acted as a cofactor in the release of 3H2O from [5-3H]dUMP, whereas no activity was detected in cytosols from TS-C1/C1. In contrast dTMP synthase activity was detected in cytosols from TS- C1/C1 cells in the presence of [6R]CH2-H4PteGlu5.(ABSTRACT TRUNCATED AT 400 WORDS)

Folate-binding protein mRNA is decreased in methotrexate-resistant KB cells.

Folate-binding protein (FBP), a high-affinity folate receptor expressed in certain malignant and normal cell lines and tissues, is responsible for cellular transport of folate and structurally related antifolates such as methotrexate in human KB (nasopharyngeal carcinoma) cells. We have developed a sensitive polymerase chain reaction (PCR) assay to quantitate folate-binding protein mRNA levels. This assay was tested in KB and methotrexate (MTX)-resistant KB (KB1BT) cell lines. Based on assay results, we conclude there are 245 molecules of folate-binding protein (FBP) mRNA per KB cell, and there is an approximately 3-fold decrease in FBP mRNA levels per KB1BT cell. The data obtained by quantitative PCR correlate well with northern blot analysis of mRNA levels and membrane-associated folate-binding protein (M-FBP) levels. Moreover, when KB1BT cells were grown in the absence of MTX for 8 months, the levels of FBP and M-FBP mRNA did not change, and there was a substantial decrease in MTX uptake, compared with KB cells.

Quantitation of dihydrofolate reductase and thymidylate synthase mRNAs in vivo and in vitro by polymerase chain reaction.

Rapid and quantitative polymerase chain reaction (PCR) assays based upon the competitive template technique have been developed for human dihydrofolate reductase (DHFR; E.C.1.5.1.3) and thymidylate synthase (TS; E.C.2.1.1.45) mRNAs. In various tumor cell lines and clinical tumor biopsies, TS mRNA levels correlated with TS levels as determined by [3H]-fluorodeoxyuridylate binding. Levels of DHFR and TS mRNAs, determined by PCR, correlated with mRNA quantitation by conventional dot blot methodology. The ratio of TS/DHFR mRNAs in a number of human carcinoma cell lines varies from 0.4 to 9.9 but ranges from 1 to greater than 1.5 x 10(3) in a number of tumor samples. Differences in the TS/DHFR mRNA ratio in tumors as compared with cultured cells reflects low levels of DHFR mRNA in some tumors. In patients treated with a combination of 5-fluorouracil and leucovorin, mRNA levels for TS increased approximately an order of magnitude in tumor samples 4 and 24 hr after drug treatment, whereas TS levels decreased. These results have significance for the biochemical pharmacology of antifolates and fluorinated pyrimidines in vivo and the relevance of cell culture models for antifolate chemotherapy and drug resistance.