Evaluation of contemporary olanzapine- and netupitant/palonosetron-containing antiemetic regimens for chemotherapy-induced nausea and vomiting.

INTRODUCTION: This post-hoc analysis retrospectively assessed data from two recent studies of antiemetic regimens for chemotherapy-induced nausea and vomiting (CINV). The primary objective was to compare olanzapine-based versus netupitant/palonosetron (NEPA)-based regimens in terms of controlling CINV during cycle 1 of doxorubicin/cyclophosphamide (AC) chemotherapy; secondary objectives were to assess quality of life (QOL) and emesis outcomes over four cycles of AC. METHODS: This study included 120 Chinese patients with early-stage breast cancer who were receiving AC; 60 patients received the olanzapine-based antiemetic regimen, whereas 60 patients received the NEPA-based antiemetic regimen. The olanzapine-based regimen comprised aprepitant, ondansetron, dexamethasone, and olanzapine; the NEPA-based regimen comprised NEPA and dexamethasone. Patient outcomes were compared in terms of emesis control and QOL. RESULTS: During cycle 1 of AC, the olanzapine group exhibited a higher rate of 'no use of rescue therapy' in the acute phase (olanzapine vs NEPA: 96.7% vs 85.0%, P=0.0225). No parameters differed between groups in the delayed phase. The olanzapine group had significantly higher rates of 'no use of rescue therapy' (91.7% vs 76.7%, P=0.0244) and 'no significant nausea' (91.7% vs 78.3%, P=0.0408) in the overall phase. There were no differences in QOL between groups. Multiple cycle assessment revealed that the NEPA group had higher rates of total control in the acute phase (cycles 2 and 4) and the overall phase (cycles 3 and 4). CONCLUSION: These results do not conclusively support the superiority of either regimen for patients with breast cancer who are receiving AC.

Cortical remodelling induced by activity of ventral tegmental dopamine neurons.

Representations of sensory stimuli in the cerebral cortex can undergo progressive remodelling according to the behavioural importance of the stimuli. The cortex receives widespread projections from dopamine neurons in the ventral tegmental area (VTA), which are activated by new stimuli or unpredicted rewards, and are believed to provide a reinforcement signal for such learning-related cortical reorganization. In the primary auditory cortex (AI) dopamine release has been observed during auditory learning that remodels the sound-frequency representations. Furthermore, dopamine modulates long-term potentiation, a putative cellular mechanism underlying plasticity. Here we show that stimulating the VTA together with an auditory stimulus of a particular tone increases the cortical area and selectivity of the neural responses to that sound stimulus in AI. Conversely, the AI representations of nearby sound frequencies are selectively decreased. Strong, sharply tuned responses to the paired tones also emerge in a second cortical area, whereas the same stimuli evoke only poor or non-selective responses in this second cortical field in naive animals. In addition, we found that strong long-range coherence of neuronal discharge emerges between AI and this secondary auditory cortical area.

Membrane-type matrix metalloproteinases in human dermal microvascular endothelial cells: expression and morphogenetic correlation.

Membrane-type matrix metalloproteinases (MT-MMP) activate the zymogen form of MMP-2/Gelatinase A on cell surfaces and are expressed in invasive tumors. We sought to identify and characterize MT-MMP in a non-malignant cell type that undergoes a physiologic and reversible invasive phenotype during angiogenesis. Human dermal microvascular endothelial cells (HDMEC) were isolated from neonatal tissue and purified by anti-CD31 (PECAM) affinity beads. MT-MMP-1 and -3 transcripts were amplified by reverse transcriptase-polymerase chain reaction and northern blots showed a single 4.5 kB mRNA for MT-MMP-1 that was modulated by angiogenic factors and phorbol ester. Immunoblotting of reduced cellular extracts with different MT-MMP-1 antibodies showed the presence of the 63-65 kDa and 57-60 kDa forms, as well as additional forms at lower molecular weights. HDMEC membranes extracted with Triton X114 were incubated with gelatin-sepharose purified MMP-2 and MMP-9 to show activation of proenzymes. Pre-incubation of HDMEC with anti-MT-MMP-1 antibodies decreased proMMP-2 conversion activity only. The movement of HDMEC and the formation of tubule-like structures in three-dimensional collagen gels was markedly delayed by preincubation with the same anti-MT-MMP-1 antibodies. These results demonstrate the presence of MT-MMP in cutaneous microvascular cells in vitro. Modulation of these cell surface proteinases by angiogenic factors, demonstration of multiple processed forms, and specific attenuation of HDMEC morphogenetic patterns in three-dimensional collagen gels implicate their potential roles in the formation of new blood vessels in the skin.

Decreased CP-1 (NF-Y) activity results in transcriptional down-regulation of topoisomerase IIalpha in a doxorubicin-resistant variant of human multiple myeloma RPMI 8226.

Decreased topoisomerase II (Topo II) activity results in resistance to antineoplastic agents targeting this enzyme. Dox1V derived from human multiple myeloma RPMI 8226 demonstrated a 4-fold resistance to doxorubicin in the absence of MDR1 overexpression or topo II mutations (Futscher B.W., Foley N., Gleason-Guzman M., Meltzer P.S., Sullivan D.M., and Dalton W.S., Int'l. J. Cancer, 66: 520-5, 1996.). Consistent with its drug resistant phenotype, a 2- to 3-fold decrease in topo II expression was identified. To investigate the molecular basis for decreased topo II expression in Dox1V, a semi-quantitative analysis of Topo II activity, protein level and mRNA transcript were performed. The results demonstrated that reduced Topo II activity is due to a decreased mRNA level. Southern blot and sequencing experiments revealed wild-type sequence of the topo II promoter in the drug resistant cells. Transient gene expression assays demonstrated that topo II is transcriptionally down-regulated in Dox1V independent of the promoter sequence of the endogenous alleles. Instead, the activity of a ubiquitous transcription factor CP-1 (NF-Y) interacting with the topo II promoter is decreased. The decrease in CP-1/NF-Y activity in Dox1V is correlated well with the decrease in topo II transcriptional activity, transcript level, Topo II protein and enzyme activity. Therefore, transcriptional down-regulation resulted from a reduced CP-1/NF-Y activity is responsible for decreased topo II expression in Dox1V cells.

DNA topoisomerase II alpha expression is associated with alkylating agent resistance.

Increased expression of DNA topoisomerase II alpha has been associated with resistance to certain DNA-damaging alkylating agents, but no causal relationship or mechanism has been established. To investigate this observation, we developed a model of topoisomerase II overexpression by transfecting a full-length Chinese hamster ovary topoisomerase II alpha into EMT6 mouse mammary carcinoma. Topoisomerase II alpha-transfected cell lines demonstrated continued topoisomerase II alpha mRNA and protein expression, which were undetectable in vector-only lines, in stationary phase (G0-G1). The topoisomerase II transfectants were approximately 5-10-fold resistant to the alkylating agents cisplatin and mechlorethamine. Upon release from G0-G1, the topoisomerase II transfectants demonstrated more rapid thymidine incorporation and shorter cell-doubling times than control cells. Purified topoisomerase II and nuclear extracts with topoisomerase II-decatenating activity bound to cisplatin-treated DNA with significantly greater affinity than to untreated DNA in a cisplatin concentration-dependent manner. These observations suggest that expression of topoisomerase II alpha may have a role in cellular resistance to antineoplastic alkylating agents. The mechanism for this may involve increased binding of topoisomerase II alpha to alkylating agent-damaged DNA.

Molecular cloning and characterization of the promoter for the Chinese hamster DNA topoisomerase II alpha gene.

To investigate the mechanisms governing the expression of DNA topoisomerase II alpha, the Chinese hamster topoisomerase II alpha gene has been cloned and the promoter region analyzed. There are several transcriptional start sites clustered in a region of 30 base pairs, with the major one being 102 nucleotides upstream from the ATG translation initiation site. Sequencing data reveal one GC box and a total of five inverted CCAAT elements (ICEs) within a region of 530 base pairs upstream from the major transcription start site. Sequence comparison between the human and Chinese hamster topoisomerase II alpha gene promoter regions shows a high degree of homology centered at the ICEs and GC box. In vitro DNase I footprinting results indicate protection by binding proteins at and around each ICE on both DNA strands. However, no obvious protection was observed for the GC box. Competition gel mobility shift assays with oligonucleotides containing either the wild-type or mutated ICE sequences suggest that identical or similar proteins specifically bind at each ICE, although with different affinities for individual ICE sequences. Chloramphenicol acetyltransferase assays employing nested 5'-deletions of the 5'-flanking sequence of the gene demonstrate that the sequence between -186 and +102, which contains three proximal ICEs, is sufficient for near wild-type level of promoter activity. When these three ICEs were gradually replaced with sequences which do not interact with the binding proteins, reducing promoter activity of the resulted constructs was observed. In conjunction with results from footprinting and gel mobility shift studies, the transient gene expression finding suggests that the ICEs are functionally important for the transcriptional regulation of the topoisomerase II alpha gene.

Conditional expression of wild-type topoisomerase II complements a mutant enzyme in mammalian cells.

Alterations in the amino acid composition, phosphorylation pattern, or intracellular levels of topoisomerase II have been associated with resistance to antineoplastic agents whose effects are mediated through interactions with this enzyme. To develop a model system with which to investigate the determinants of topoisomerase II sensitivity or resistance to antineoplastic agents that target this enzyme, a cDNA encoding the wild-type Drosophila melanogaster topoisomerase II was ligated into a mammalian expression vector containing a glucocorticoid-inducible mouse mammary tumor virus promoter and transfected into an epipodophyllotoxin-resistant Chinese hamster ovary cell line (VPM(r)-5). In two transfectants carrying an intact, full-length Drosophila topoisomerase II cDNA, exposure to the inducing agent, dexamethasone (10 microM), resulted in complementation of the endogenous mutant topoisomerase II and phenotypic reversion to etoposide sensitivity. In the presence of glucocorticoid, etoposide-induced cytotoxicity increased 20-fold, despite the fact that Drosophila topoisomerase II mRNA expression was only 0.1% of that of the endogenous mammalian topoisomerase II. Induced cells demonstrated a marked increase in DNA single strand breaks compared with uninduced resistant cells, thereby providing biochemical evidence supporting increased DNA strand cleavage due to activation of the Drosophila enzyme. These observations demonstrate the ability of a wild-type Drosophila topoisomerase II to complement a mutant mammalian enzyme and suggest that transfectants capable of conditional topoisomerase II expression represent a useful model for studies of the biochemical pharmacology and structure-function relationships of normal and mutant enzymes.

Molecular cloning and identification of a point mutation in the topoisomerase II cDNA from an etoposide-resistant Chinese hamster ovary cell line.

Topoisomerase II (Top II) is the target enzyme for many antineoplastic drugs such as epipodophyllotoxins, anthracyclines, and acridines. Cell lines with alterations in Top II are resistant to drugs that interact with the enzyme. Studies of the Top II from a Chinese hamster ovary line, VpmR-5, that is resistant to VP-16 and VM-26, demonstrated that it is very similar, qualitatively and quantitatively, to its normal counterpart except that DNA cleavage by the VpmR-5 enzyme is not stimulated by VP-16 or VM-26. To understand the basis for the drug-resistant phenotype, the Top II cDNAs were isolated from both Chinese hamster ovary (CHO) and VpmR-5 cells by cDNA cloning with lambda gt22, and the entire cDNAs were sequenced. A mutation of G-->A at nucleotide 1478 was the only alteration observed in the VpmR-5 Top II cDNA compared with the wild-type gene. The mutation in VpmR-5 was confirmed by sequencing DNA fragments amplified from the genomic DNA by the polymerase chain reaction. Southern blot hybridization analysis of genomic DNA demonstrated loss of a Top II allele in VpmR-5 probably occurred during the development of resistance to etoposide. The mutation in VpmR-5 changes amino acid 493 from arginine to glutamine and is located adjacent to a putative ATP binding site of Top II. Mutations in an analogous region have been identified in two human leukemia cell lines by amplification of segments of Top II cDNA with Taq DNA polymerase. Taken together, these observations suggest that mutations in this region of the gyrase B domain of mammalian topoisomerase II may be capable of conferring resistance to antineoplastic agents that interact with this enzyme.

Oncogenes result in genomic alterations that activate a transcriptionally silent, dominantly selectable reporter gene (neo).

Although oncogenes and tumor suppressor genes have been implicated in carcinogenesis and tumor progression, their relationship to the development of genomic instability has not been elucidated. To examine this role, we transfected oncogenes (polyomavirus middle [Py] and large T [MT and LT]) and adenovirus serotype 5 E1A) into two NIH 3T3-derived cell lines, EN/NIH 2-4 and EN/NIH 2-20. Both cell lines contain two stable integrants of a variant of the retrovirus vector pZipNeoSV(x)1 that has been modified by deletion of the enhancer elements from the long terminal repeats. DNA rearrangements activating the silent neomycin phosphotransferase gene (neo) present in these integrants were identified by selection of cells in the antibiotic G418. Whereas control-transfected EN/NIH cell lines do not yield G418-resistant subclones (GRSs), a fraction of oncogene-transfected EN/NIH 2-4 (8 of 19 Py MT, 5 of 17 Py LT, and 11 of 19 E1A) and 2-20 (7 of 15 Py MT) cell lines gave rise to GRSs at differing frequencies (0.33 x 10(-6) to 46 x 10(-6) for line 2-4 versus 0.11 x 10(-6) to 1.3 x 10(-6) for line 2-20) independent of cell generation time. In contrast, a distinctly smaller fraction of mutant Py MT-transfected EN/NIH cell lines (1 of 10 MT23, 1 of 10 MT1015, and 0 of 10 MT59b) resulted in GRSs. Southern analysis of DNA from selected oncogene-transfected GRSs demonstrated genomic rearrangements of neo-containing cellular DNA that varied in type (amplification and/or novel fragments) and frequency depending on the specific oncogene and EN/NIH cell line used in transfection. Furthermore, only one of the two neo-containing genomic loci present in both EN/NIH cell lines appeared to be involved in these genomic events. In addition to effects related to the genomic locus, these observations support a role for oncogenes in the development of genetic changes associated with tumor progression.

Production and use of nonradioactive hybridization probes.

Molecular hybridization is a useful technique for identifying specific target sequences even when they are present as a single copy in a complex population. It can be performed either on a solid matrix on which pure DNA (or RNA) is bound (blot hybridization) or on tissue sections (in situ hybridization). Until recently, the probes used in hybridization were usually labeled with radioisotopes. However, the short half-life, disposal, and safety problems of radioactive probes stimulated the development of nonradioactive hybridization techniques. In these, the probes are labeled with nonradioactive reporter molecules, which can be haptens, proteins, digoxigenin, biotin, and so forth. These reporter molecules can then be detected by enzyme-labeled antibodies or streptavidin (in the case of biotinylated probes). Of these reporter molecules, biotin and digoxigenin have several advantages over the others because of their small size. Therefore, they minimally interfere with hybridization efficiency. In addition, the high affinity of the binding of biotin and streptavidin (K ( d ) = 10(-15) M) is almost equivalent to covalent bonds. In fact, the biotin system was the first nonradioactive hybridization technique sensitive enough for routine use on Mot hybridization (1) and in situ hybridization (2).

Production and use of nonradioactive hybridization probes.

Molecular hybridization is a useful technique for identifying specific target sequences even when they are present as a single copy in a complex population. It can be performed either on a solid matrix on which pure DNA (or RNA) is bound (blot hybridization) or on tissue sections (in situ hybridization). Until recently, the probes used in hybridization were usually labeled with radioisotopes. However, the short half-life, disposal, and safety problems of radioactive probes stimulated the development of nonradioactive hybridization techniques. In these, the probes are labeled with nonradioactive reporter molecules, which can be haptens, proteins, digoxigenin, biotin, and so forth. These reporter molecules can then be detected by enzyme-labeled antibodies or streptavidin (in the case of biotinylated probes). Of these reporter molecules, biotin and digoxigenin have several advantages over the others because of their small size. Therefore, they minimally interfere with hybridization efficiency. In addition, the high affinity of the binding of biotin and streptavidin (K ( d ) = 10(-15) M) is almost equivalent to covalent bonds. In fact, the biotin system was the first nonradioactive hybridization technique sensitive enough for routine use on Mot hybridization (1) and in situ hybridization (2).

Parvovirus as a cause of hydrops fetalis: detection by in situ DNA hybridisation.

Lung tissue from 13 cases of unexplained non-immunological hydrops fetalis was examined by in situ hybridisation to detect parvovirus. Four specimens contained parvovirus DNA in cells in the blood vessel lumina and alveoli. Twenty six control cases were negative for parvovirus DNA. As there was no known epidemic of parvovirus infection during the study period, this suggests that parvovirus is a relatively common cause of non-immunological hydrops fetalis. In situ hybridisation may have a role in clinical medicine, particularly for retrospective investigations.

Simultaneous extraction from clinical biopsies of high-molecular-weight DNA and RNA: comparative characterization by biotinylated and 32P-labeled probes on Southern and Northern blots.

A method for efficient simultaneous extraction of high-molecular-weight DNA and RNA from solid mammalian tissues including clinical biopsies is described. It is based on the disruption and subsequent melting of deep frozen tissue in the presence of frozen phenol and nucleic acid extraction buffer; this allows for simultaneous disruption of tissue and inactivation of nucleases. The yield is about 0.7-5.8 mg of DNA and 0.5-8.1 mg of total RNA/g of tissue depending upon the tissue type; this is higher than the yield of other methods tested. Analysis of total RNA by denaturing gel electrophoresis, and of DNA and poly(A)+ RNA by Southern and Northern blot hybridization using 32P and biotinylated probes, indicated that c-Ha-ras gene and its transcripts were undegraded. Biotinylated and 32P probes had approximately the same sensitivity in detecting nucleic acids on Southern and Northern blots. This extraction procedure is simple and, when used with biotinylated probes, is rapid, inexpensive, and nonhazardous. The methodology can be modified for use with other clinical samples and cells grown in culture.

The role of vitamin A in the glycosylation reactions of glycoprotein synthesis in an 'in vitro' system.

Microsomal membrane preparations from rat livers, when incubated with labelled sugar-nucleotides, were shown to synthesize labelled oligosaccharide-lipids in the presence of excess exogenous dolichyl phosphate. Under the incubation conditions defined in the present study, dolichyl pyrophosphoryl(DolPP)GlcNAc2-Man5, DolPPGlcNAc2Man9 and DolPPGlcNAc2Man9Glc3 were the principal oligosaccharide-lipids formed by both control and vitamin A-deficient membranes. However, deficient membranes synthesized 3.2 +/- 0.8 times as much oligosaccharide-lipids and 2.6 +/- 0.7 times as much dolichyl phosphate mannose (DolPMan) and dolichyl phosphate glucose (DolPGlc) as the controls. The transfer of the oligosaccharide chain from the dolichol carrier to the endogenous protein acceptors in vitamin A-deficient microsomes (microsomal fractions) was only 57.5 +/- 9.5% of that of controls. After endo-beta-N-acetylglucosaminidase treatment, only one oligosaccharide species was isolated from both control and vitamin A-deficient microsomal glycoproteins, and was characterized as GlcNAcMan9Glc3. We conclude that the decreased incorporation of labelled mannose and glucose from sugar-nucleotides into the glycoproteins must be due to decreased transfer of GlcNAc2Man9Glc3 from the dolichol carrier to the protein acceptors. This conclusion was further substantiated by the finding that control membranes transferred 4-6 times as much labelled oligosaccharides from exogenously added dolichol-linked substrate (DolPPGlcNAc2Man9Glc3) to endogenous microsomal protein acceptors as compared with the vitamin A-deficient membranes. Attempts to reverse this defect by addition of retinol or retinyl phosphate (a source of retinyl phosphate mannose) to the incubations were unsuccessful.

Cellular oncogenes in neoplasia.

In recent years cellular homologues of many viral oncogenes have been identified. As these genes are partially homologous to viral oncogenes and are activated in some tumour cell lines they are termed "proto-oncogenes". In tumour cell lines proto-oncogenes are activated by either quantitative or qualitative changes in gene structure: activation of these genes was originally thought to be a necessary primary event in carcinogenesis, but activated cellular oncogenes, unlike viral oncogenes, do not transform normal cells in culture. In experimental models cooperation between two oncogenes can induce transformation of early passage cells, and this has become the basis of an hypothesis for multistep carcinogenesis. Proto-oncogene products also show sequence homology to various components in the mitogenic pathway (growth factors, growth factor receptors, signal transducing proteins and nuclear proteins), and it has been postulated that they may cause deregulation of the various components of this pathway. In human tumours single or multiple oncogene activation occurs. The pattern of oncogene activation in common solid malignancies is not consistent within any one class of tumour, nor is it uniform between classes, with three exceptions. In neuroblastoma, breast cancer, and perhaps in lung cancer there is relatively consistent activation of N-myc, neu, and c-myc/N-myc, respectively. Amplification of these genes generally correlates with poor prognosis. The introduction of methods for the direct study of oncogene transcription and their products will undoubtedly broaden our vision of cancer biology in man and, hopefully, add diagnostic and prognostic precision to tumour typing.

Detection of sub-picogram quantities of specific DNA sequences on blot hybridization with biotinylated probes.

A sensitive method for detecting biotinylated DNA probes on dot and Southern blots is described which is based on the principle outlined by Leary et al (1). This system has two main components: detection of biotinylated DNA by a two-step procedure with streptavidin and poly(alkaline phosphatase); and blocking background with Tween 20. 32fg and 80fg of lambda phage DNA was detected on dot and Southern blot hybridizations respectively. 150fg of beta-globin was detected on Southern blots of genomic DNA. This method is fast, reproducible and can detect single copy genes in 0.25 micrograms genomic DNA on Southern blots.

Sensitive system for visualising biotinylated DNA probes hybridised in situ: rapid sex determination of intact cells.

A fast and sensitive method for detecting biotinylated deoxyribonucleic acid (DNA) probes was used for sex determination of cells and tissues by in situ hybridisation of a probe "specific" for the Y chromosome (pHY 2.1). Within 24 hours this procedure visualizes the Y chromosome in fetal and adult cells and tissue, without background noise. This procedure should facilitate antenatal determination of sex on small numbers of uncultured cells. The sensitivity of the procedure also permits the chromosomal assignment of genes present in low copy number.