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The dihydrofolate reductase (DHFR) gene (dhfr) promoter contains &-acting elements for the transcription factors Spl and E2F. Given the ability of Spl to activate the dhfr promoter, we have evaluated the contribution of Spl to the cell-growth regulation of the dhfr gene. Using gel-mobility assays performed with DNA probes from the minimal promoter of the hamster dhfr gene and nuclear extracts from cultured hamster cells (CHO K1) we show that the binding of Spl to the dhfr promoter is cell-growthphase regulated. Accordingly, dhfr transcription and mRNA levels in K1 cells increase upon serum stimulation. Cytological detection of Spl by immunofluorescence reveals a decrease of this protein in the process leading to the GO state, and an increase upon serum stimulation of quiescent cells. These results were confirmed by western blot analysis. It is concluded that Spl progressively binds to the hamster dhfr promoter after stimulation of cell proliferation, which can account for the transcriptional regulation of the dhfr gene during the cell cycle. The role of Spl in the specific control of dhfr during the cell cycle was confirmed in vivo using cell lines derived from dhfr-negative cells transfected with dhfr plasmids carrying either the wild-type or mutated Spl-binding or E2F-binding sites in the dhfr minimal promoter.Keywords: Spl, dihydrofolate reductase; proliferation; cell-cycle; transcription.Dihydrofolate reductase (DHFR) catalyzes the synthesis of purines, thymidylate and glycine, and is therefore needed for the replication of DNA in the S phase of the cell cycle. Although transcription of the dhfr gene increases dramatically at the G1/ S boundary [I-31, dhfr mRNA is present throughout the cell cycle [ 1, 41, accounting for a steadily increasing DHFR enzymic activity [5]. Considerable efforts have been made to identify the constitutive factors that control the dhfr promoter, and the factors that confer cell-cycle regulation on this gene. In this regard, the dhfr promoter contains cis-acting elements for the transcription factors Spl and E2F. It has been demonstrated that distinct GC boxes, responsible for Spl binding, direct transcription at two principal transcriptional initiation sites in rodents [6, 71. Only the most proximal GC box, at least in hamster cells, is needed to correctly initiate transcription at the major proximal site (minimum promoter). Deletion of this GC box abolishes transcription of the dhfr gene [8], despite the presence of the E2F-binding element just downstream of the major transcriptional start. It has also been shown that E2F from human cells (HeLa or HEL), either in nuclear extracts or purified, is able to bind to its recognition sequence in the murine [9] and hamster [lo, 111 dhfr genes, and that the E2F site is required for growth regulation 112, 131. Given that E2F can be bound by the tumor suppressor and cell-cycle regulator retinoblastoma protein (Rb) [14-171 these findings support a model for dhfr gene expression in which hypophosphorylated Rb represses E2F in resting cells, t...

The dihydrofolate reductase (dhfr) promoter is powerfully activated by the transcription factor Sp1. It has been suggested that Sp1 is a potential target for transcriptional regulation by the cell cycle regulator retinoblastoma protein (Rb), and so we have explored this possibility using the hamster dhfr gene as a model. By the use of DNA probes from the hamster dhfr gene promoter, containing the most proximal GC box (minimal promoter), and nuclear extracts from cultured hamster cells (CHO K1), we show that polyclonal and monoclonal antibodies against Rb supershift the binding of Sp1. Nuclear extract immunoprecipitation with antiRb followed by Western analysis using anti-Sp1 also shows that Rb is complexed to Sp1. Complementary Immunoprecipitation/WB analysis shows both forms of Rb protein in the anti-Sp1 immunoprecipitates. Moreover, nuclear extract immunodepletion of Rb abolishes Sp1 gel-shift. The interaction between Rb and Sp1 is maintained in all the phases of the cell cycle. Transient overexpression of Rb in dhfr negative cells co-transfected with a dhfr minigene driven by its minimal promoter increases DHFR activity and potentiates transcription when overexpressing Sp1. Both e ects are severely reduced when the co-transfections are performed with a homologous dhfr minigene containing a single point mutation in the GC box. Thus, the activation by Rb of the dhfr gene may be exerted through Sp1. Stable transfectants of pCMVRb in K1 cells show an increase in both mRNA and DHFR activity. It is concluded that Sp1 is physically associated with Rb, and that this association increases Sp1-mediated transcription of the hamster dhfr gene.

Background: Accurate characterization of the infection stage in parvovirus B19(B19V)-positive plasma donations would help establish the donation deferral period to contribute to a safe fractionation pool of plasma. Methods: Viral DNA load of 74 B19V DNA-positive plasma samples from whole blood donations was determined by titration using nucleic acid testing. Markers of cellular (neopterin) and humoral (B19V-specific IgM and IgG) immune response were determined by ELISA in 32 B19V DNA-positive samples and in 13 B19V DNA-negative samples. The infection progression profile was estimated according to B19V DNA load and the presence of immune response markers. Results: B19V DNA load in the 74 samples was 10⁶-10¹³ IU/ml. The distribution of 14 out of 32 selected B19V DNA-positive samples plus 2 B19V DNA-negative samples with no immune response marker followed along an upward curve according to B19V DNA load. After the peak, the distribution of 18 immune marker-positive samples followed along a downward curve according to their B19V DNA load and was grouped as follows: neopterin (n = 4), neopterin+ IgM (n = 8), neopterin + IgM + IgG (n = 3), IgM + IgG (n = 2), IgM (n = 1). There were 11 B19V DNA-negative IgG-positive samples. Conclusion: This study of B19V-DNA load and levels of neopterin, IgM, and IgG allows for reliable characterization and distribution into the different stages of B19V infection.

We examined the effect of suboptimal concentrations of cyclin-dependent kinase inhibitors, which do not interfere with cell proliferation, on retinoblastoma expression in hamster (Chinese hamster ovary K1) and human (K562 and HeLa) cells. To achieve this, we used the chemical inhibitors roscovitine and olomoucine (which inhibit CDK2 preferentially), UCN-01 (which also inhibits CDK4/6) and p21 (as an intrinsic inhibitor). All chemical inhibitors and overexpression of p21 strongly induced retinoblastoma protein expression. UCN-01-mediated retinoblastoma expression was caused by an increase in both the levels of retinoblastoma mRNA and the stability of the protein. The expression of the transcription factor Sp1, a retinoblastomainteracting protein, was also enhanced by all the cyclin-dependent kinase inhibitors tested. However, Sp1 expression was caused by an increase in the levels of Sp1 mRNA without modification in the stability of the protein. By using luciferase experiments, the transcriptional activation of both retinoblastoma and Sp1 promoters by UCN-01 was confirmed. Bisindolylmaleimide I, at concentrations causing a similar or higher inhibition of protein kinase C than UCN-01, provoked a lower activation of retinoblastoma and Sp1 expression. Finally, the effects of cyclin-dependent kinase inhibitors on dihydrofolate reductase gene expression were evaluated. Treatment with UCN-01 increased cellular dihydrofolate reductase mRNA levels, and dihydrofolate reductase enzymatic activity was enhanced by UCN-01, roscovitine, olomoucine and p21, in transient transfection experiments. These results support a mechanism for the self-regulation of retinoblastoma expression, and point to the need to establish the appropriate dose of cyclin-dependent kinase inhibitors as antiproliferative agents in anticancer treatments.Keywords: retinoblastoma gene product; Sp1; UCN-01; roscovitine; dihydrofolate reductase.Cyclin-dependent kinases (CDKs) are key regulators of cell cycle progression. They constitute the catalytic subunits of holoenzymes formed in combination with regulatory subunits named cyclins. Thirteen CDKs [1,2] and at least 25 cyclins [2] have been reported to date. Cyclin expression varies during the cell cycle and the cyclin/CDK holoenzyme is activated by phosphorylation of specific residues in the CDK catalytic subunit by the cdk-activating kinase [3,4]. CDKs are involved in transcriptional control [5], mitotic progression [6], DNA repair (CDK7) [7], differentiation of brain neurons (CDK5) [8] and play a crucial role in the progression of cells from G1 to S phase by regulating the phosphorylation state of the retinoblastoma gene product (Rb). The tumor suppressor Rb is a nuclear protein of 928 amino acids [9] that is present in distinct phosphorylation states depending on the phase of the cell cycle [10,11]: it is nonphosphorylated when newly synthesized; hypophosphorylated in early G1; and hyperphosphorylated in late G1, S, and G2/M phases. In mitosis, a protein phosphatase 1-like protein removes all phosphates f...

We analyzed the differential gene expression in the pancreatic cancer cell line NP-18 upon induction of apoptosis caused by cyclin-dependent kinase inhibition triggered by either overexpression of the tumor suppressor gene p16^INK4Ausing an adenoviral construction or incubation with the chemical inhibitors, roscovitine or olomoucine. Screening was performed using cDNA arrays from Clontech that allowed the determination of the expression of 1,176 genes specifically related with cancer. The analysis was carried out using the Atlas Image 2.01 (Clontech) and GeneSpring 4.2 (Silicon Genetics) softwares. Among the differentially expressed genes, we chose for further validation histone deacetylase 1 (HDAC1), von Hippel Lindau and decorin as upregulated genes, and Sp1, hypoxia-inducible factor-1 alpha and DNA primase as downregulated genes. The changes in the expression of these genes to mRNA were validated by quantitative RT-PCR and the final translation into protein by Western blot analysis. Inhibition of HDAC activity, Sp1 binding and DNA primase expression led to an increase in the level of apoptosis, both in parental cells and in doxorubicin-resistant cells. Therefore, these proteins could constitute possible targets to develop modulators in cancer chemotherapy that would increase or restore apoptosis.

RNA-based arbitrarily primed PCR (RAP-PCR) was used to identify sequences in CHO K1 cells that were differentially expressed upon methotrexate incubation during the development of resistance to this drug. Ten different RAP products were isolated, cloned and sequenced. Among these, we identified one sequence that showed 84% identity with the nucleotide sequence of rat cytochrome c oxidase subunit II, and 90% identity with the amino acid sequence of this protein. This RAP fragment was up-regulated in a dose- and time-dependent manner. The overexpression of cytochrome c oxidase subunit II mRNA as a result of methotrexate incubation was corroborated by quantitative RT-PCR and Northern blot analysis. Incubation of cells with sodium azide, a specific cytochrome c oxidase inhibitor, decreased the number of resistant colonies after methotrexate treatment. Thus, overexpression of cytochrome c oxidase is involved in the development of resistance to methotrexate. These results suggest that sodium azide may be used as a modulator in chemotherapy with methotrexate.

The effect of incubations with anti‐sense phosphorothioate oligonucleotides directed toward sequences of dihydrofolate reductase (DHFR) RNA has been tested on Chinese hamster ovary cells. The selected targets were the 5'‐untranslated region, the translational start, the splice sites and branch point of intron 1 and polyadenylation regions 1 and 3 of the DHFR RNA. To introduce the oligonucleotides, the cationic liposome DOTAP was used. The oligonucleotides most effective at causing cytotoxicity were ATNL and DTNL, both directed toward the translation‐start site, at a range of concentrations between 1 and 4 μM. The minimum time for the oligonucleotide to exert its full cytotoxic effect was 3 days. Excess of oligonucleotide diminished the cytotoxic effect. Oligonucleotide uptake was monitored by the incorporation of [32P]‐ or fluorescein‐labeled oligonucleotide and was found to depend on liposome and oligonucleotide concentrations and duration of incubation. Formation of in vitro complexes between the oligonucleotide and the liposome was also studied. Cytotoxicity was observed when the oligonucleotide was incubated with cell lines containing either the endogenous gene or co‐transfected DHFR minigenes. Cell incubation with ATNL caused a time‐dependent decrease in the levels of DHFR mRNA and enzymatic activity. Moreover, a cell line bearing amplification at the dhfr locus was equally affected by the action of ATNL. Human hepatoma cells were also affected by treatment with the counterpart of ATNL in the human DHFR mRNA sequence. Our results set the basis for a possible cancer therapy with anti‐sense oligonucleotides using DHFR as the target. Int. J. Cancer 81:785–792, 1999. © 1999 Wiley‐Liss, Inc.