Elevated serum Her-2/neu level predicts decreased response to hormone therapy in metastatic breast cancer.

PURPOSE: To determine the effect of elevation of serum HER-2/neu on response to hormone therapy. PATIENTS AND METHODS: Seven hundred nineteen metastatic patients with estrogen receptor-positive (ER(+)), progesterone receptor-positive, or both or ER status unknown breast cancer were randomized in three independent clinical trials to receive second-line hormone therapy with either megestrol acetate or an aromatase inhibitor (fadrozole or letrozole). An automated enzyme-linked immunosorbent assay specific for the extracellular domain of the HER-2/neu (c-erbB-2) oncoprotein product was used to detect serum levels. RESULTS: Two hundred nineteen patients (30%) had elevated serum HER-2/neu protein levels, using the mean + 2 SD (15 ng/mL) from the serum of healthy women as an upper limit. Response to treatment was available for 711 patients. The response rate (complete responses plus partial responses plus stable disease) to endocrine therapy was 45% in 494 patients with non-elevated and 23% in 217 patients with elevated serum HER-2/neu levels (P <.0001). Median duration of treatment response (using the time to progression [TTP] variable for patients who responded) was shorter in the group with elevated serum HER-2/neu levels (11.7 months) compared with the patient group with non-elevated levels (17.4 months). TTP, time to treatment failure, and median survival (17.2 months v 29.6 months) were also significantly shorter in the patients with elevated serum HER-2/neu levels (P <.0001). CONCLUSION: Patients with ER(+) and serum HER-2/neu-positive metastatic breast cancer are less likely to respond to hormone treatment and have a shorter duration of response than ER(+) and serum HER-2/neu-negative patients. Their survival duration is also shorter.

A novel bispecific antisense oligonucleotide inhibiting both bcl-2 and bcl-xL expression efficiently induces apoptosis in tumor cells.

Bcl-2 and Bcl-xL are inhibitors of apoptosis frequently overexpressed in solid tumors. The bcl-2 and bcl-xL mRNAs share a region of homology comprising nucleotides 605-624 and 687-706, respectively, which differs by only three nucleotides. This sequence does not occur in the proapoptotic splice variant bcl-xS. To test the possibility that oligonucleotides targeting this region have the potential to down-regulate bcl-2 and bcl-xL expression simultaneously, three 2'-O-methoxy-ethoxy-modified phosphorothioate oligonucleotides were designed. These oligonucleotides differed in the number of mismatches to bcl-2 and bcl-xL and in the number of nucleotides to which the modifications were made. The effects of these oligonucleotides on bcl-2 and bcl-xL expression, as well as their abilities to induce apoptosis, were assessed in small cell and non-small cell lung cancer cell lines expressing different basal levels of bcl-2 and bcl-xL. Although all oligonucleotides down-regulated bcl-2 and bcl-xL expression, oligonucleotide 4625, which has no mismatching nucleotides to bcl-2 but three to bcl-xL, two of which were modified by 2'-O-methoxy-ethoxy residues, showed the strongest bispecific activity on the transcript and protein level. In all cell lines this bispecific activity induced apoptotic cell death, as demonstrated by increased uptake of propidium iodide, a 10-100-fold increase in caspase-3-like protease activity, and nuclear condensation and fragmentation. This is the first report of a bcl-2/bcl-xL bispecific antisense oligonucleotide that deserves attention as a therapeutic compound in lung cancer and other malignancies in which bcl-2 and/or bcl-xL are overexpressed.

Change in lactate production in Myc-transformed cells precedes apoptosis and can be inhibited by Bcl-2 overexpression.

As a result of Myc-dependent transcription of the LDH-A gene, Myc-transformed cells (Rat1-Myc) exhibit increased lactate production rates (LPR) even under aerobic conditions (the Warburg effect). Recently, the increased susceptibility to stress-induced apoptosis associated with Myc transfection has been linked to the overexpression of the LDH-A gene. In this report we demonstrate that the overexpression of the anti-apoptotic protein Bcl-2 in Rat1-Myc cells (Rat1-Myc-Bcl-2) reduces the molar ratio of lactate production to glucose consumption (Y(L/G)). The Bcl-2 induced reduction in Y(L/G) may be associated with reduced expression of the LDH-A gene, or a decrease in LDH-A activity. Stimulation of apoptosis by staurosporine, a protein kinase C inhibitor, reduces the LPR in Rat1-Myc cells in a dose-dependent manner. The staurosporine effect on the LPR is rapid and precedes the execution phase of apoptosis as defined by caspase activation and PARP cleavage. This effect on LPR is completely blocked by Bcl-2 overexpression. Serum starvation alone does not affect the LPR of Rat1-Myc or Rat1-Myc-Bcl-2 cells; however, the effect of staurosporine on the LPR of Rat1-Myc cells is potentiated by serum starvation. These data demonstrate that Bcl-2 overexpression reduces the Y(L/G) in Rat1-Myc cells, perhaps via a reduction in the activity or expression of the LDH-A gene, and this reduction may desensitize cells to some pro-apoptotic stimuli. The reduction in LPR in response to staurosporine may be an early step in the induction of apoptosis in Rat1-Myc cells. By abolishing the reduction in LPR, Bcl-2 may protect Rat1-Myc cells from staurosporine-induced apoptosis. Moreover, the lack of effect by serum starvation on the LPR supports a model in which serum starvation induces apoptosis through a pathway distinct from that of the staurosporine and glucose-dependent apoptotic pathway(s) in Myc-transformed cells.

Ras-dependent apoptosis correlates with persistent activation of stress-activated protein kinases and induction of isoform(s) of Bcl-x.

Ras proteins are signal transducers for many cellular responses. However, it is not well established whether Ras-signaling also contributes to apoptosis. We have constructed H-RasR12-transformed Rat1 fibroblasts using tetracycline operator/repressor (TetO/TetR)-based conditional vectors. Rat1/TetO-RasR12 (Rat1-Ras) cells produced high levels of H-RasR12 protein and exhibited oncogenic transformation. Treatment of Rat1-Ras cells with 0.1% serum triggered massive apoptosis. Rat1-Ras cells expressed increased basal activities of extracellular response kinase (ERK) and p46/p54 stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK). Interestingly, Ras-dependent apoptosis correlated with further persistent activation of both p46 and p54 SAPK/JNK and concurrent inhibition of ERK. Differential modulation of SAPK/JNK and ERK was not detected in tetracycline-treated cells that did not commit apoptosis. Furthermore, two Bcl-x related proteins of 15 kDa and 18 kDa were highly induced in apoptotic Rat1-Ras cells. Our results establish a direct role for Ras in apoptosis, and suggest a functional relationship between H-Ras, SAPK/JNK, ERK and Bcl-x in regulating apoptosis.

Identification of a cyclin-cdk2 recognition motif present in substrates and p21-like cyclin-dependent kinase inhibitors.

Understanding how cyclin-cdk complexes recognize their substrates is a central problem in cell cycle biology. We identified an E2F1-derived eight-residue peptide which blocked the binding of cyclin A and E-cdk2 complexes to E2F1 and p21. Short peptides spanning similar sequences in p107, p130, and p21-like cdk inhibitors likewise bound to cyclin A-cdk2 and cyclin E-cdk2. In addition, these peptides promoted formation of stable cyclin A-cdk2 complexes in vitro but inhibited the phosphorylation of the retinoblastoma protein by cyclin A- but not cyclin B-associated kinases. Mutation of the cyclin-cdk2 binding motifs in p107 and E2F1 likewise prevented their phosphorylation by cyclin A-associated kinases in vitro. The cdk inhibitor p21 was found to contain two functional copies of this recognition motif, as determined by in vitro kinase binding/inhibition assays and in vivo growth suppression assays. Thus, these studies have identified a cyclin A- and E-cdk2 substrate recognition motif. Furthermore, these data suggest that p21-like cdk inhibitors function, at least in part, by blocking the interaction of substrates with cyclin-cdk2 complexes.

Apoptosis research enters the ICE age.

Recent elucidation of the structure of interleukin-1 beta-converting enzyme (ICE), a protease with sequence homology to a nematode protein associated with programmed cell death, opens a new chapter in the study of how proteases may control cellular suicide.

Inhibition of type 1 human immunodeficiency virus replication by a tat antagonist to which the virus remains sensitive after prolonged exposure in vitro.

The transactivator of transcription, Tat, of human immunodeficiency virus type 1 (HIV-1) is required for viral replication. Inhibition of Tat function could have the potential to keep integrated provirus in dormancy. In the presence of Tat, Ro 24-7429, an analog of Ro 5-3335, inhibited expression of indicator genes controlled by the HIV-1 long terminal repeat promoter in transient transfection assays and in a constitutive cell line at noncytotoxic concentrations. Reduction of steady-state mRNA of the indicator gene by the compound correlated with reduction of the gene product in the constitutive cell line. Ro 24-7429 has broad activity against several strains of HIV-1 in different cell lines, peripheral blood lymphocytes, and macrophages (IC90 = 1-3 microM). Importantly, Ro 24-7429 inhibited viral replication in both acute and chronic infection in vitro, a characteristic expected of a Tat antagonist and not shared by viral reverse transcriptase inhibitors. Consistent with this, the compound reduced cell-associated viral RNA and proteins and partially restored cell-surface CD4 in chronically infected cells. After 2 years of continued weekly passage of the virus in fresh CEM cells grown in the presence of the compound at 1 or 10 microM, the virus did not develop resistance to the drug. These results indicate that the compound's action might involve a cellular factor.

Characterization of recombinant HIV-1 Tat and its interaction with TAR RNA.

Recombinant HIV-1 Tat (Tat 1-86) has been purified from the cytoplasmic fraction of Escherichia coli without the use of protein denaturants or chaotropic agents. Chloroquine-mediated uptake of the purified protein into cells resulted in transactivation of the HIV LTR promoter. Tat retains 1.64 mol of Zn2+/mol of protein by atomic absorption spectroscopy. Circular dichroism measurements indicated that the structure of recombinant Tat contains 15-20% alpha-helix. Filter binding assays showed that Tat binds to a 63-nucleotide target TAR RNA with a dissociation constant (Kd) of 10 nM at 25 degrees C, 0.05 M ionic strength, pH 7.5, in a 1:1 Tat-TAR RNA stoichiometry. Nonelectrostatic interactions provide the principal source of free energy of association. While the pH optimum occurs over a wide H+ concentration, the salt dependence of Kd indicates formation of a single ion pair. UV-induced protein-RNA cross-linking produced a labeled Tat-TAR RNA adduct, indicating that direct contact occurred between the Tat protein and TAR RNA.

Purification and characterization of recombinant Rev protein of human immunodeficiency virus type 1.

Recombinant Rev protein of human immunodeficiency virus type 1 has been expressed in Escherichia coli and purified by ion-exchange and gel-filtration chromatography. Specific binding of the purified protein to the Rev-responsive element of the viral RNA is demonstrated. Physical characterization of the purified protein by circular dichroism and intrinsic fluorescence spectroscopy indicate that the protein preparation is suitable for structural analysis. Circular dichroism measurements show that the protein is approximately 40-45% alpha-helix. Tryptophan fluorescence measurements suggest that the single tryptophan residue is located near the surface of the protein. Gel-filtration chromatography of the protein indicates that it has an apparent molecular mass of 33,000 daltons. This suggests that the protein in solution forms a stable tetramer consisting of monomers having molecular mass of 13,000 daltons.

Interaction of the human immunodeficiency virus type 1 Rev protein with a structured region in env mRNA is dependent on multimer formation mediated through a basic stretch of amino acids.

Interaction of the human immunodeficiency virus type 1 (HIV-1) Rev protein with a structured region within env mRNA (termed RRE) mediates the export of virus structural mRNAs from the nucleus to the cytoplasm. We show that the region encompassing the basic stretch of amino acids is essential for the ability of Rev to bind to RRE RNA and function in vivo. By use of a functional truncated Rev protein in conjunction with authentic Rev, effects on gel mobilities of the Rev-RRE RNA complex attributable to multimerization of Rev protein were observed. Rev proteins, unable to multimerize, failed to bind RRE RNA. Identification of Rev mutants capable of forming multimers, but unable to bind RRE RNA, suggests that the multimerization and RNA-binding domains can be distinguished and that multimerization is likely a prerequisite for formation of the RRE RNA-binding site. A mutant Rev protein, shown previously to function as a trans-dominant inhibitor of Rev function, bound to RRE RNA as a multimer to a similar extent as wild-type Rev. This observation is consistent with the hypothesis that regulation of HIV gene expression by Rev involves the interaction with cellular factors and that the trans-dominant Rev is probably defective in this function.

The 3'-terminal sequence of a wound tumor virus transcript can influence conformational and functional properties associated with the 5'-terminus.

We recently reported the presence of segment-specific inverted repeats within the terminal regions of wound tumor virus genomic segments (J. V. Anzol, Z. Xu, T. Asamizu, and D. L. Nuss, 1987, Proc. Natl. Acad. Sci. USA, 84, 8301-8305). This report describes a series of experiments designed to investigate potential intramolecular interactions involving the 5'- and 3'-terminal domains of wound tumor virus transcripts. A series of transcription vectors were constructed which allowed the synthesis of an exact copy of the transcript corresponding to genomic segment S8 and four analogs that differed from the authentic sequence only at the immediate 3'-terminus. Modifications designed to extend or alter the 3'-terminal inverted repeat altered the in vitro translational efficiency of the transcript and the sensitivity of phosphodiester bonds within the immediate 5'-terminal domain to digestion by nuclease T1. These results were consistent with computer-assisted secondary structure analyses of the complete nucleotide sequence of transcripts corresponding to six genomic segments which predicted intramolecular interactions involving the terminal inverted repeats. Potential roles of the terminal domains in expression, sorting and packaging of a segmented RNA genome are considered.

In vitro methylation of the 5'-flanking regions of the mouse beta-globin gene.

The enzymatic methylation of the 5'-flanking region of the mouse beta-globin (major) gene containing putative regulatory regions has been investigated. In vitro methylation of this 368-base pair regulatory DNA by a DNA methyltransferase obtained from mouse erythroleukemia cells yields an asymmetric methylation pattern. Of the 10 available CG pairs, only 5-6 are modified, leading to one hemimethylated site and two apparently fully methylated sites. Only CG pairs which are localized in a 29-base pair cluster are methylated. The data suggest that a CG cluster approximately 100 base pairs upstream from the CAP site may be the in vivo site of methylation in the 5'-regulator region of the mouse beta-globin gene.

Primary DNA sequence determines sites of maintenance and de novo methylation by mammalian DNA methyltransferases.

Analysis of the enzymatic methylation of oligodeoxynucleotides containing multiple C-G groups showed that hemimethylated sites in duplex oligomers are not significantly methylated by human or murine DNA methyltransferase unless those sites are capable of being methylated de novo in the single- or double-stranded oligomers. Thus, the primary sequence of the target strand, rather than the methylation pattern of the complementary strand, determines maintenance methylation. This suggests that de novo and maintenance methylation are the same process catalyzed by the same enzyme. In addition, the study revealed that complementary strands of oligodeoxynucleotides are methylated at different rates and in different patterns. Both primary DNA sequence and the spacing between C-G groups seem important since in one case studied, maximal methylation required a specific spacing of 13 to 17 nucleotides between C-G pairs.

DNA methylation: sequences flanking C-G pairs modulate the specificity of the human DNA methylase.

Synthetic single-stranded oligodeoxynucleotides of known sequence have been used as in vitro substrates for a partially purified HeLa cell DNA methylase. Although most oligonucleotides tested cannot be used by the HeLa DNA methylase in vitro, we have found a unique 27mer, containing 2 C-G pairs, that is an excellent substrate for the enzyme. Analysis of the methylation of the 27mer, its derivatives and other oligomer substrates reveal that the HeLa DNA methylase does not significantly methylate an oligomer which contains just one C-G pair. In addition, only one of the two C-G pairs in the 27mer is methylated and this methylation is abolished if the other C-G pair is converted to a C-A pair. Furthermore, the HeLa enzyme apparently cannot methylate C-G pairs located in compounds containing a high A + T content. The most efficient methylation occurs with multiple separated C-G pairs in a compound with a high G + C content (greater than 65%). The results suggest that clustering of C-G pairs in regions of the DNA high in G + C content may be the preferred site for DNA methylation in vivo.

Selective modification of an alpha subunit of chloroplast coupling factor 1.

Lucifer yellow (4-amino-N-[3-(vinylsulfonyl)phenyl]naphthalimide-3,6-disulf onate), a fluorescent probe that can react covalently with sulfhydryl or amino groups, has been used to modify chloroplast coupling factor 1 (CF1). Conditions are described under which Lucifer yellow selectively labels the alpha subunit of CF1 to the extent of about 1 mol of probe per mole of CF1. An especially reactive amino group is apparently labeled, and modification has little effect on the ATPase activity of the enzyme. Lucifer yellow is a useful probe for fluorescence energy transfer measurements. The distances between this probe and fluorescent and absorbing molecules attached to seven specific sites on the beta, gamma, and epsilon subunits were determined. These distances converge to a single location. In addition to providing further information about the structure of CF1, these results suggest that the alpha subunits of CF1 are not structurally equivalent.

The effect of flanking sequences on the de novo methylation of C-G pairs by the human DNA methylase.

The HeLa DNA methylase can methylate selected cytosine residues in oligodeoxynucleotides as small as 12-16 nucleotides in length in vitro. The maximum methylation rate seems to require oligomers having more than one C-G in the molecule even when only one of the C-G pairs is methylated. Compounds which contain a high G+C content also seem to be favored substrates. The use of defined synthetic oligodeoxynucleotides permits one to demonstrate that flanking DNA sequences can be critical in determining whether a C-G site can be methylated.

Role of a disulfide bond in the gamma subunit in activation of the ATPase of chloroplast coupling factor 1.

The relationship between activation of the latent ATPase activity of isolated chloroplast coupling factor 1 (CF1) and reduction of a disulfide in the gamma subunit has been assessed. The sulfhydryl residues involved in the disulfide bond are distinct from residues normally accessible to maleimide modification during incubation of thylakoids in the dark or the light. Dithiothreitol-induced activation is time dependent, and correlates with reduction of the disulfide. Sulfhydryl residues exposed during activation can be reoxidized to disulfide by incubation with iodosobenzoate , with a concomitant loss of ATPase activity. Activation and deactivation are reversible, but deactivation is prevented by treatment of the reduced enzyme with N-ethylmaleimide. Heat activation does not reduce the disulfide bond unless dithiothreitol is present during activation. Prior heating of CF1, which partially activates the enzyme, renders the disulfide more susceptible to subsequent dithiol reduction. The activity obtained when heat and dithiothreitol are used together is approximately equal to the sum of the partial activations obtained with heat or dithiothreitol alone. Iodosobenzoate has no effect on heat-activated CF1. Enzyme activated by heating in the presence of dithiothreitol can be partially deactivated, consistent with reversal of the activity attributable to the dithiol effect. Fluorescence polarization of anilinonaphthylmaleimide bound to the reduced enzyme indicates that the sulfhydryl residues involved in the disulfide are in a less rigid environment than the other two sulfhydryl residues in the gamma subunit. Polarization of anilinonaphthylmaleimide bound to these sulfhydryls is reduced by heat treatment of CF1. The increased susceptibility of the disulfide to reduction upon heat treatment, and the activation of ATPase activity with or without disulfide bond cleavage are indicative of conformational changes within the gamma subunit that occur during the conversion of CF1 from a latent to an active ATPase. In addition the results are consistent with at least two distinct conformational forms of CF1 that can hydrolyze ATP.

Selective modification of coupling factor 1 in spinach chloroplast thylakoids by a fluorescent maleimide.

N-(1-Anilinonaphthyl-4)maleimide (ANM) has been used to modify coupling factor 1 (CF1), the terminal coupling factor of photophosphorylation in chloroplasts. As with other monofunctional maleimides, incubation of thylakoids with ANM in the light, but not in the dark, causes energy transfer inhibition of photophosphorylation. In the dark, sites on both the gamma and epsilon subunits of CF1 are modified. The light-accessible site is also on the gamma subunit. Trypsin digestion of the enzyme after dithiothreitol activation reveals that the dark-and light-accessible sites on the gamma subunit are different amino acid residues. Fluorescence of ANM bound at the dark-and light-accessible sites has been measured after isolation of CF1 from thylakoids. The fluorescence emission maximum of ANM at the light-accessible site is blue-shifted and the quantum yield is increased 2-fold relative to ANM bound at dark-accessible sites. On the soluble enzyme, fluorescence polarization is high and equivalent for ANM bound at both dark-and light-accessible sites. Fluorescence energy transfer from a tryptophan in a hydrophilic region of the epsilon subunit to ANM bound to the epsilon subunit but not to the gamma subunit has been observed. The significance of these observations is discussed with respect to the structure of the gamma subunit and its role in conformational transitions within CF1 that occur during energization of the membrane.

Adenine nucleotide binding sites on beef heart F1-ATPase. Specificity of cooperative interactions between catalytic sites.

Cooperative interactions between nucleotide binding sites on beef heart mitochondrial F1-ATPase have been studied by measuring substrate-promoted release of 5'adenylyl-beta,gamma-imidodiphosphate (AMP-PNP) from a single high affinity site. The site is initially loaded by incubating F1 with an equimolar amount of the nonhydrolyzable ATP analog. When unbound [3H]AMP-PNP is removed and the complex diluted to a concentration below the Kd, release of ligand shows an apparent absolute requirement for medium ADP. Release is biphasic with the extent of release during the initial rapid phase dependent on the concentration of medium ADP. Although phosphate alone has no effect, it enhances the rapid phase of ADP-promoted release over 2-fold with a half-maximal effect at 60 micrometers P1. The binding of efrapeptin (A23871) to the F1.AMP-PNP complex completely prevents ADP-promoted dissociation. Although AMP-PNP release also occurs in the presence of medium ATP, the F1.AMP-PNP complex does not dissociate if an ATP-regenerating system of sufficient capacity to prevent accumulation of medium ADP is added. Consistent with an inability of nucleoside triphosphate to promote release is the failure of medium, nonradioactive AMP-PNP to affect retention of the 3H-labeled ligand. The stability of F1.AMP-PNP complex in the absence of medium nucleotide and the highly specific ability of ADP plus P1 to promote rapid release of the ATP analog are interpreted as support for an ATP synthesis mechanism that requires substrate binding at one catalytic site for product release from an adjacent interacting site.