A phase I/II prospective, single arm trial of gefitinib, trastuzumab, and docetaxel in patients with stage IV HER-2 positive metastatic breast cancer.

Inhibition of the HER-2 pathway via the monoclonal antibody trastuzumab has had a major impact in treatment of HER-2 positive breast cancer, but de novo or acquired resistance may reduce its effectiveness. The known interplay between the epidermal growth factor receptor (EGFR) and HER-2 receptors and pathways creates a rationale for combined anti-EGFR and anti-HER-2 therapy in HER-2 positive metastatic breast cancer (MBC), and toxicities associated with the use of multiple chemotherapeutic agents together with biological therapies may also be reduced. We conducted a prospective, single arm, phase I/II trial to determine the efficacy and toxicity of the combination of trastuzumab with the EGFR inhibitor gefitinib and docetaxel, in patients with HER-2 positive MBC. The maximum tolerated dose (MTD) was determined in the phase I portion. The primary end point of the phase II portion was progression-free survival (PFS). Immunohistochemical analysis of biomarker expression of the PKA-related proteins cAMP response element-binding protein (CREB), phospho-CREB and DARPP-32 (dopamine and cAMP-regulated phosphoprotein of 32 kDa) plus t-DARPP (the truncated isoform of DARPP-32); PTEN; p-p70 S6K; and EGFR was conducted on tissue from metastatic sites. Nine patients were treated in the phase I portion of the study and 22 in the phase II portion. The MTD was gefitinib 250 mg on days 2-14, trastuzumab 6 mg/kg, and docetaxel 60 mg/m(2) every 21 days. For the 29 patients treated at the MTD, median PFS was 12.7 months, with complete and partial response rates of 18 and 46%, and a stable disease rate of 29%. No statistically significant correlation was found between response and expression of any biomarkers. We conclude that the combination of gefitinib, trastuzumab, and docetaxel is feasible and effective. Expression of the biomarkers examined did not predict outcome in this sample of HER-2 overexpressing metastatic breast cancer.

Precise localization of m6A in Rous sarcoma virus RNA reveals clustering of methylation sites: implications for RNA processing.

N6-methyladenosine (m6A) residues are present as internal base modifications in most higher eucaryotic mRNAs; however, the biological function of this modification is not known. We describe a method for localizing and quantitating m6A within a large RNA molecule, the genomic RNA of Rous sarcoma virus. Specific fragments of 32P-labeled Rous sarcoma virus RNA were isolated by hybridization with complementary DNA restriction fragments spanning nucleotides 6185 to 8050. RNA was digested with RNase and finger-printed, and individual oligonucleotides were analyzed for the presence of m6A by paper electrophoresis and thin-layer chromatography. With this technique, seven sites of methylation in this region of the Rous sarcoma virus genome were localized at nucleotides 6394, 6447, 6507, 6718, 7414, 7424, and 8014. Further, m6A was observed at two additional sites whose nucleotide assignments remain ambiguous. A clustering of two or more m6A residues was seen at three positions within the RNA analyzed. Modification at certain sites was found to be heterogeneous, in that different molecules of RNA appeared to be methylated differently. Previous studies have determined that methylation occurs only in the sequences Gm6AC and Am6AC. We observed a high frequency of methylation at PuGm6ACU sequences. The possible involvement of m6A in RNA splicing events is discussed.

Use of a cloned multidrug resistance gene for coamplification and overproduction of major excreted protein, a transformation-regulated secreted acid protease.

Malignantly transformed mouse fibroblasts synthesize and secrete large amounts of major excreted protein (MEP), a 39,000-dalton precursor to an acid protease (cathepsin L). To evaluate the possible role of this protease in the transformed phenotype, we transfected cloned genes for mouse or human MEP into mouse NIH 3T3 cells with an expression vector for the dominant, selectable human multidrug resistance (MDR1) gene. The cotransfected MEP sequences were efficiently coamplified and transcribed during stepwise selection for multidrug resistance in colchicine. The transfected NIH 3T3 cell lines containing amplified MEP sequences synthesized as much MEP as did Kirsten sarcoma virus-transformed NIH 3T3 cells. The MEP synthesized by cells transfected with the cloned mouse and human MEP genes was also secreted. Elevated synthesis and secretion of MEP by NIH 3T3 cells did not change the nontransformed phenotype of these cells.

Effect of carbohydrate position on lysosomal transport of procathepsin L.

To study the role of carbohydrate in lysosomal protein transport, we engineered two novel glycosylation signals (Asn-X-Ser/Thr) into the cDNA of human procathepsin L, a lysosomal acid protease. We constructed six mutant cDNAs encoding glycosylation signals at mutant sites Asn-138, Asn-175, or both sites together, in the presence or absence of the wild-type Asn-204 site. We stably transfected wild-type and mutant cDNAs into NIH3T3 mouse fibroblasts and then used species-specific antibodies to determine the glycosylation status, phosphorylation, localization, and transport kinetics of recombinant human procathepsin L containing one, two, or three glycosylation sites. Both novel glycosylation sites were capable of being glycosylated, although Asn-175 was utilized only 30-50% of the time. Like the wild-type glycosylation at Asn-204, carbohydrates at Asn-138 and Asn-175 were completely sensitive to endoglycosidase H, and they were phosphorylated. Mutant proteins containing two carbohydrates were capable of being delivered to lysosomes, but there was not a consistent relationship between the efficiency of lysosomal delivery and carbohydrate content of the protein. Pulse-chase labeling revealed a unique biosynthetic pattern for proteins carrying the Asn-175 glycosylation sequence. Whereas wild-type procathepsin L and mutants bearing carbohydrate at Asn-138 appeared in lysosomes by about 60 min, proteins with carbohydrate at Asn-175 were processed to a lysosome-like polypeptide within 15 min. Temperature shift, brefeldin A, and NH4Cl experiments suggested that the rapid processing did not occur in the endoplasmic reticulum and that Asn-175 mutants could interact with the mannose 6-phosphate receptor. Taken together, our results are consistent with the interpretation that Asn-175 carbohydrate confers rapid transport to lysosomes. We may have identified a recognition domain in procathepsin L that is important for its interactions with the cellular transport machinery.

Magnetic-affinity cell sorting of human multidrug-resistant cells.

We have developed a method for isolating multidrug-resistant cells from a heterogeneous cell population using the magnetic-affinity cell-sorting system. Human KB carcinoma cell lines expressing different amounts of P-glycoprotein have been selected by use of the monoclonal antibody MRK-16 coupled to magnetic particles. This specific, rapid, and sensitive method allows the selection of viable and clonable drug-resistant cells from various drug-resistant human and murine cell populations. This method may prove useful in isolating drug-resistant cells from tumors with heterogeneous P-glycoprotein expression for further analysis.

Use of the stress-inducible grp78/BiP promoter in targeting high level gene expression in fibrosarcoma in vivo.

Current advances in human gene therapy open up new frontiers for molecular therapies of cancer. However, one major limitation in cancer gene therapy is the lack of a general tumor-specific promoter which allows stringent and high level expression of the therapeutic reagent in malignantly transformed but not normal tissues. Hallmark features of solid tumors such as glucose deprivation, chronic anoxia, and acidic pH induce the glucose-regulated proteins, in particular, GRP78/BiP, a M(r) 78,000 endoplasmic reticulum-localized protein with chaperone and calcium-binding properties. We report here that a truncated rat grp78 promoter with most of the distal basal elements removed can be utilized as a potent internal promoter in a retroviral vector to drive high level expression of a reporter gene in a murine fibrosarcoma model system. The stress-inducible grp78 promoter offers a novel approach for gene delivery systems targeting transcription in tumorigenic cells.

Transduction of NIH 3T3 cells with a retrovirus carrying both human MDR1 and glutathione S-transferase pi produces broad-range multidrug resistance.

In the experiments, we examined the ability of a retroviral vector, pHaMASV, to encode two potential chemoprotective genes on separate transcription units. We previously described the pHaMSV vector, which includes the human MDR1 gene as a selectable marker and chemoprotective gene, plus an internal SV40 promoter for expressing a second heterologous gene along with MDR1 [M. E. Metz, D. M. Best, and S. E. Kane. Virology, 208: 634-643, 1995]. To test the ability of this vector to deliver two therapeutic genes simultaneously, the cDNA for human glutathione S-transferase pi (GST pi, the most abundant member of the glutathione S-transferase family in human tumor cells) was inserted into pHaMASV, and this plasmid was transfected into ecotropic packaging cells. The resulting pHaMASV.GST pi ecotropic retrovirus, which was produced at a titer of 2 x 10(6) colony-forming units/ml, was used to transduce NIH 3T3 cells. After initial selection in 60 ng/ml colchicine, a population of transduced cells was exposed to stepwise increasing colchicine concentrations to select for amplified expression of MDR1. As MDR1 expression increased, the expression of GST pi increased in concert, as demonstrated by Northern analysis, Western analysis, and measurement of glutathione S-transferase activity. Transduced cells growing in 1280 ng/ml colchicine had about 3-fold higher total glutathione S-transferase activity than nontransduced cells and 2.5-fold higher activity than transduced cells growing in 60 ng/ml colchicine. Northern hybridizations demonstrated a 3-5-fold increase in both the full-length retroviral message encoding MDR1 and the subgenomic mRNA encoding GST pi after amplification of resistance from 60 to 1280 ng/ml colchicine. The cytotoxic effects of several xenobiotics were evaluated in NIH 3T3 cells transfected with MDR1 (3T3.MDR) or transduced with the MDR1-GST pi retrovirus (3T3.GST640 or 3T3.GST1280) to evaluate the ability of our vector to produce a spectrum of drug resistances specific for the genes expressed. 3T3.MDR and 3T3.GST1280 cells expressing equivalent levels of MDR1 had identical levels of resistance to doxorubicin or colchicine. These results suggest that GST pi expression did not contribute to doxorubicin resistance in this model system. However, 3T3.GST640 cells were about 4-fold resistant to ethacrynic acid and 1-chloro-2,4-dinitrobenzene compared to cells expressing MDR1 alone, consistent with the ability of GST pi to conjugate both of these cytotoxins. Increases in drug resistance paralleled increases in gene-specific mRNA and recombinant protein levels in all cases.4+ chemotherapy.

Desensitization and sensitization of cells to fluoropyrimidines with different antisenses directed against thymidylate synthase messenger RNA.

Previous studies have shown that the cytotoxicity of fluoropyrimidines is mediated, in large part, by inhibition of the enzyme thymidylate synthase (TS). The aim of this study was to determine whether the chemosensitivity of human cancer cells to fluoropyrimidines could be increased by decreasing TS expression with antisense oligodeoxyribonucleotides (ODNs). ODNs (18-mers) targeted at the AUG translational initiation site of TS mRNA inhibited translation in a sequence- and dose-dependent manner in a rabbit reticulocyte lysate in vitro translation system. Treatment of human colon cancer HT-29 cells with antisense ODNs decreased TS catalytic activity in the cells in a dose-dependent manner over a short period, but the longer-term effect of the TS antisense ODN treatment was actually to increase the amount of TS in the cells and to decrease their sensitivity to 5-fluoro-2'-deoxyuridine (FdUrd). However, when human nasopharyngeal cancer KB31 cells were transfected with a plasmid (pHaMAGRP) construct containing the TS antisense fragment (+ 1 to + 422) under the control of a glucose-regulated promoter, the expression of both TS protein and TS catalytic activity was decreased by nearly 30% (P = 0.014), and sensitivity of these cells to FdUrd was enhanced by approximately 8-fold (P = 0.021). No changes in the levels of expression of TS protein or FdUrd-associated cytotoxicity were observed in control, vector-transfected cells. No change was observed in the sensitivity of transfected cells toward either cisplatin or Adriamycin. These results show that the level of expression of TS in human malignant cells can be down-regulated with antisense TS RNA, and their sensitivity to fluoropyrimidines can, thereby, be increased.

Comparison of the breadth and complexity of bovine viral diarrhea (BVDV) populations circulating in 34 persistently infected cattle generated in one outbreak.

Exposure to bovine viral diarrhea viruses (BVDV) results in acute and persistent infections. Persistent infections result from in utero exposure during the first trimester of gestation. Clinical presentation, in persistently infected cattle (PI), is highly variable. The reasons for this variation is largely unknown. The BVDV circulating in PI exist as quasispecies (swarms of individual viruses). An outbreak resulting in 34 PI cattle presented an opportunity to compare a large number of PI׳s. Methods were developed to compare the circulating viral populations within PI animals. It was found that PI animals generated in the same outbreak carry circulating viral populations that differ widely in size and diversity. Further, it was demonstrated that variation in PI viral populations could be used as a quantifiable phenotype. This observation makes it possible to test the correlation of this phenotype to other phenotypes such as growth rate, congenital defects, viral shed and cytokine expression.

A new vector using the human multidrug resistance gene as a selectable marker enables overexpression of foreign genes in eukaryotic cells.

A new vector, pSK1.MDR, has been constructed for expressing nonselectable genes in eukaryotic cells. The vector uses the human multidrug resistance gene, MDR1, as a dominant selectable marker and contains an additional transcription unit plus a unique SalI cloning site for inserting nucleotide sequences to be expressed. To test this expression system, a cDNA (IL2R) for the 55-kDa interleukin-2 receptor was inserted into the SalI site, and the resulting plasmid was transfected into NIH3T3 cells. Cells which acquired the MDR1 gene were selected with colchicine, and cells with high levels of MDR1 expression were selected by growth in increasing concentrations of the drug. Drug resistant cells also expressed the cotransferred, nonselected IL2R gene, and its expression was increased to 740,000 receptors per cell by growing cells in high concentrations of colchicine. The MDR1 system represents a very efficient method for synthesizing large amounts of protein in a wide variety of eukaryotic cells.

MDR1 bicistronic vectors: analysis of selection stringency, amplified gene expression, and vector stability in cell lines.

The human multidrug resistance-1 gene (MDR1) is a dominant selectable and amplifiable marker in mammalian tissue culture cells. MDR1 is also being investigated as a gene therapy tool, both to protect normal cells against chemotherapy-related toxicity and to serve as an in vivo selectable marker for the overexpression of non-selectable therapeutic genes. The success of these strategies will depend on whether MDR1 expression can be sustained at levels high enough to confer a survival advantage on target cells. However, the MDR1 selection system is quite stringent, requiring high gene expression for transduced cells to survive in the presence of drug. The current report is a detailed molecular analysis of MDR1 selection stringency compared with the common neo selectable marker. A bicistronic vector encoding MDR1 and neo genes linked through an internal ribosome entry site was transferred into NIH 3T3 mouse fibroblasts and K562 human leukemia cells; cells were then exposed to colchicine (to select for MDR1 expression) or to G418 (to select for neo expression). Surviving populations and individual clones of cells were analyzed for expression levels of MDR1 and neo gene products; resistance to colchicine, paclitaxel, and G418; level and integrity of bicistronic mRNA; and structural integrity, integration number, and copy number of vector DNA. These studies provide direct evidence that colchicine selection is more stringent than G418 selection; that increased selection pressure with colchicine leads to increased gene expression; that increased gene expression can be accommodated primarily by gene amplification, even within an individual transduced clone and starting from a single-copy proviral integration event; and that the clonal diversity of a transduced population of cells is influenced significantly by the stringency of selection. Taken together, these results have important implications for the potential utility of MDR1 as a selectable marker and as a gene therapy tool in hematopoietic cells.

HydroLink gel electrophoresis (HLGE). II. Applications of a new polymer matrix to dsDNA analysis.

HydroLink materials represent a novel family of gels composed of unique polymer matrices. The applications of HydroLink to molecular biology and, specifically, to DNA technology have been carefully investigated. Our results indicate that the HydroLink matrix developed for double-stranded DNA (dsDNA) is an excellent tool for electrophoretic separations in fixed electric fields. Excellent linear resolution from 100 to 5000 base pairs is easily achieved with good resolution albeit non-linear from 6000 to 23000 base pairs. The broad range of separation in addition to increased mechanical strength of dsDNA HydroLink represents a distinct advantage over other matrices currently used in DNA electrophoretic analysis.

Incidence and clinicopathologic correlation of fetal vessel thrombosis in mono- and dichorionic twin placentas.

OBJECTIVE: To determine the incidence of fetal vessel thrombosis in monochorionic-diamniotic and dichorionic-diamniotic twin placentas, and its association with intrauterine growth retardation (IUGR), hypertensive disorders of pregnancy, twin-twin transfusion syndrome (TTTS), fetal vascular anastomoses, chorangiosis, and chorioamnionitis. STUDY DESIGN: Histologic slides from 80 pairs of monochorionic and 80 pairs of dichorionic twin placentas were reviewed for evidence of fetal vessel thrombosis (≥5 adjacent avascular terminal villi with upstream intravascular fibrin thrombi). Associations with clinical and other pathologic variables were analyzed by χ(2) tests. RESULT: Thrombosis occurred in 7.5% of monochorionic and 3.1% of dichorionic twin placentas (P=0.090). It was associated with IUGR among the monochorionic twins (P=<0.0001) and with hypertensive disorders of pregnancy among the dichorionic twins (P=0.018). Vascular anastomoses, TTTS, chorangiosis, and chorioamnionitis were not associated with fetal vessel thrombosis. CONCLUSION: Fetal vessel thrombosis was identified more frequently in monochorionic twins, but this difference was not statistically significant. It is associated with IUGR in monochorionic twins, but not with TTTS or fetal vascular anastomoses at the chorionic surface.

Mouse procathepsin L lacking a functional glycosylation site is properly folded, stable, and secreted by NIH 3T3 cells.

The role of glycosylation in the synthesis, transport, and localization of procathepsin L has been analyzed. Procathepsin L is the precursor form of a lysosomal cysteine protease which is synthesized in large amounts by malignantly transformed cells and cells treated with growth factors or tumor promoters. As for other lysosomal hydrolases, procathepsin L is predominantly secreted when it is overexpressed, despite the presence of the lysosomal sorting marker mannose 6-phosphate. To study the role of glycosylation in procathepsin L trafficking, we constructed a cDNA in which the codon for Asn-204 was mutated to encode Gln. When this mutated cDNA was transfected into NIH 3T3 cells, a completely nonglycosylated form of procathepsin L was expressed. The protein appeared to be folded properly, as judged by its ability to be proteolytically processed to a lower molecular weight form during incubation at pH 4. Nonglycosylated protein was stable, both as unprocessed proenzyme and after processing at pH 4, and it was predominantly secreted instead of being delivered to lysosomes. Nonglycosylated and endogenous glycosylated procathepsin L were secreted by NIH 3T3 cells with identical kinetics. These studies therefore confirm that Asn-204 is the normal functional carbohydrate acceptor in procathepsin L and that carbohydrate in wild-type procathepsin L serves predominantly as a lysosomal targeting signal, with little or no role in protein folding or stability. Furthermore, there does not appear to be a major mannose 6-phosphate-independent route of lysosomal localization in NIH 3T3 cells. In experiments with a second mutant, the glycosylation signal at Asn-251, which normally is not utilized, was capable of serving as a carbohydrate acceptor, suggesting that there is normally a structural impediment to glycosylation at Asn-251 in procathepsin L.

Inhibition of methylation at two internal N6-methyladenosine sites caused by GAC to GAU mutations.

We previously have mapped N6-methyladenosine (m6A) sites within the genomic RNA of Rous sarcoma virus (RSV). The results of that study and of experiments using inhibitors of methylation suggest that m6A might be involved in mRNA processing events. We describe an approach for directly analyzing the function of m6A in RNA and for studying the sequence specificity of the m6A methylase. Two sites of methylation in RSV (nucleotides 7414 and 7424) were altered by oligonucleotide-directed mutagenesis. The highly conserved GAC consensus sequence at those sites was changed to GAU. The new sequences were no longer methylated in the RSV genomic RNA; the GAC sequence was required for efficient base modification at those two adenosines. The altered m6A pattern did not affect viral RNA processing or the viral life cycle within infected cells.

The role of cathepsin L in malignant transformation.

Procathepsin L is the major excreted protein of malignantly transformed mouse fibroblasts and it is one of the major acid cysteine proteases in mammalian cells. The broad general protease activity of cathepsin L against a variety of extracellular matrix proteins as well as other intracellular protein substrates, and its regulation by factors which stimulate growth, suggest that it contributes to the transformed phenotype. Possible functions in antigen processing, invasiveness, metastasis, and turnover of intracellular and secreted proteins involved in growth regulations have been proposed.

Basic fibroblast growth factor sensitizes NIH 3T3 cells to apoptosis induced by cisplatin.

One mechanism by which chemotherapeutic agents kill tumor cells is by induction of apoptosis. Basic fibroblast growth factor (bFGF/FGF-2) has been reported to inhibit apoptosis in NIH 3T3 cells treated with chemotherapy drugs. We have investigated how bFGF modulates apoptosis induced by cisplatin in NIH 3T3 cells. Treatment with 10 microgram/ml cisplatin for 12 h induced apoptosis in 2 to 13% of the cells at 24 h post-treatment. Preincubation with 10 ng/ml bFGF for 24 h led to cisplatin-induced apoptosis in 20% to 50% of the cells. Preincubation with lower concentrations of bFGF (0.1-1 ng/ml) or simultaneous addition of bFGF and cisplatin had no effect on the amount of apoptosis. Pretreatment with bFGF also significantly decreased the dose-dependent survival of NIH 3T3 cells exposed to cisplatin, as determined by colony formation. Cells treated with 10 ng/ml bFGF showed a distinct morphology, appearing smaller and more refractile, before cisplatin exposure. The enhancement of cisplatin-induced apoptosis and the morphology shift demonstrated the same dose response to bFGF, and both effects were reversible if bFGF was removed from the medium for 24 h before cisplatin treatment. Mitogenic response to bFGF by NIH 3T3 cells saturated at 0.5 ng/ml, as measured by (3)H-thymidine uptake, and this response was blocked by coaddition of suramin, an inhibitor of FGF ligand-receptor interactions. Suramin did not reverse the enhancement of cisplatin-induced apoptosis by bFGF. Therefore, bFGF sensitized NIH 3T3 cells to cisplatin, and this effect might be mediated through a pathway separate from that used for mitogenic signaling.