Maria G. Pallavicini

β1 Integrin Inhibitory Antibody Induces Apoptosis of Breast Cancer Cells, Inhibits Growth, and Distinguishes Malignant from Normal Phenotype in Three Dimensional Cultures and In vivo

Current therapeutic approaches to cancer are designed to target molecules that contribute to malignant behavior but leave normal tissues intact. beta(1) integrin is a candidate target well known for mediating cell-extracellular matrix (ECM) interactions that influence diverse cellular functions; its aberrant expression has been implicated in breast cancer progression and resistance to cytotoxic therapy. The addition of beta(1) integrin inhibitory agents to breast cancer cells at a single-cell stage in a laminin-rich ECM (three-dimensional lrECM) culture was shown to down-modulate beta(1) integrin signaling, resulting in malignant reversion. To investigate beta(1) integrin as a therapeutic target, we modified the three-dimensional lrECM protocol to approximate the clinical situation: before treatment, we allowed nonmalignant cells to form organized acinar structures and malignant cells to form tumor-like colonies. We then tested the ability of beta(1) integrin inhibitory antibody, AIIB2, to inhibit tumor cell growth in several breast cancer cell lines (T4-2, MDA-MB-231, BT474, SKBR3, and MCF-7) and one nonmalignant cell line (S-1). We show that beta(1) integrin inhibition resulted in a significant loss of cancer cells, associated with a decrease in proliferation and increase in apoptosis, and a global change in the composition of residual colonies. In contrast, nonmalignant cells that formed tissue-like structures remained resistant. Moreover, these cancer cell-specific antiproliferative and proapoptotic effects were confirmed in vivo with no discernible toxicity to animals. Our findings indicate that beta(1) integrin is a promising therapeutic target, and that the three-dimensional lrECM culture assay can be used to effectively distinguish malignant and normal tissue response to therapy.

Molecular cytogenetic abnormalities in multiple myeloma and plasma cell leukemia measured using comparative genomic hybridization.

Comparative genomic hybridization (CGH) was used to identify recurrent regions of DNA sequence loss and gain in 21 multiple myeloma (MM) and plasma cell leukemia (PCL) primary tumor specimens and cell lines. Multiple regions of non‐random sequence loss and gain were observed in 8/8 primary advanced stage tumors and 13/13 cell lines. Identification of sequence copy number changes was facilitated by statistical analyses that reduce subjectivity associated with identification of copy number changes and by requiring that sequence changes are visible using both red‐ and green‐labeled tumor DNA. Loss of sequence on 13q and 14q and gain of sequence on 1q and chromosome 7 occurred in 50–60% of the population. In general, cell lines carry more and larger regions of sequence gain and loss than primary tumors. Regions of sequence copy number change that recur among MM cell lines and primary tumors include, in order of prevalence, enh(1q12qter), dim(13), enh(7), enh(3q22q29), enh(11q13.3qter), dim(14q11.2q31), enh(8q21qter), enh(3p25pter), dim(17p11.2p13), and dim(6q22.1q23). Population distributions of genome‐wide changes in primary tumors reveal “hot‐spots” of sequence loss from 13q12.1‐q21, 13q32‐q34, 14q11.2‐q13, and 14q23‐q31. Genomic changes detected using CGH are consistent with those identified using banding analyses, although recurrent involvement of additional regions of the genome are also evident. A higher prevalence of genomic changes is visible using CGH compared to banding. Identification of recurrent regions of sequence gain and loss provides opportunities to identify regions of the genome that may be involved in the malignant phenotype and/or disease progression. Genes Chromosom. Cancer 19:124–133, 1997.

Effects of methotrexate on transfected DNA stability in mammalian cells.

The chromosomal locations, amounts, and level of expression of transfected, amplified c-myc and dihydrofolate reductase sequences were measured in cells cultured in the presence and absence of methotrexate. These studies show that the location and amount of transfected sequences, as well as the level of expression, were more variable when the cells were cultured in methotrexate.

A t-Statistic for Objective Interpretation of Comparative Genomic Hybridization (CGH) Profiles

An objective method for interpreting comparative genomic hybridization (CGH) is described and compared with current methods of interpretation. The method is based on a two-sample t-statistic in which composite test:reference and reference:reference CGH profiles are compared at each point along the genome to detect regions of significant differences. Composite profiles are created by combining CGH profiles measured from several metaphase chromosomes for each type of chromosome in the normal human karyotype. Composites for both test:reference and reference:reference CGH analyses are used to generate mean CGH profiles and information about the variance therein. The utility of the method is demonstrated through analysis of aneusomies and partial gain and loss of DNA sequence in a myeloid leukemia specimen. Banding analyses of this specimen indicated inv (3)(q21q26), del (5)(q2?q35), -7, +8 and add (17)(p11.2). The t-statistic analyses of CGH data indicated rev ish enh (8) and rev ish dim (5q31.1q33.1,7q11.23qter). The undetected gain on 17p was small and confined to a single band (17p11.2). Thus, the t-statistic is an objective and effective method for defining significant differences between test and reference CGH profiles.

Heterogeneity in the angiogenic response of a BT474 human breast cancer to a novel vascular endothelial growth factor‐receptor tyrosine kinase inhibitor: Assessment by voxel analysis of dynamic contrast‐enhanced MRI

To investigate the heterogeneity in the angiogenic response of a human breast cancer xenograft to a novel vascular endothelial growth factor (VEGF)‐receptor tyrosine kinase inhibitor, AG‐013736, using dynamic contrast‐enhanced MR imaging (DCE‐MRI).

Cell cycle analysis using flow cytometry.

This manuscript reviews the utility of flow cytometry for the study of cell proliferation. The applications of univariate DNA distribution analysis to cytokinetic studies of asynchronous and perturbed cell populations are discussed briefly. The newly developed technique for simultaneous flow cytometric measurement of cellular DNA content and amount of incorporated bromodeoxyuridine is discussed in more detail. The cytochemistry required for this analysis is reviewed as are its applications to: determination of the fractions of cells in the G1-, S- and G2 + M-phases of the cell cycle; determination of the G1-, S- and G2 + M phase durations and dispersions and growth fraction for asynchronous cells; detection of ara-C resistant cells present at low frequency in an otherwise sensitive population; and analysis of the cytokinetic response of a solid murine tumour to treatment in vivo with a cell cycle specific agent.

Enhanced pharmacodynamic and antitumor properties of a histone deacetylase inhibitor encapsulated in liposomes or ErbB2-targeted immunoliposomes.

ErbB2-overexpressing human cancers represent potentially sensitive targets for therapy by candidate histone deacetylase (HDAC) inhibitors as we have shown that HDAC inhibitors can selectively reduce ErbB2 expression by repressing the ErbB2 promoter and accelerating the decay of cytoplasmic ErbB2 transcripts. To extend these in vitro findings and enhance the in vivo pharmacodynamic properties of HDAC inhibitors, we stably encapsulated a potent hydroxamate-based HDAC inhibitor (LAQ824) within long-circulating liposomes (Ls-LAQ824) and immunoliposomes (ILs-LAQ824) bearing >10,000 LAQ824 molecules per nanovesicle. Liposomal LAQ824 exhibits prolonged in vivo stability and, unlike free LAQ824, circulates with a half-life of 10.8 hours following a single i.v. injection. Three weekly i.v. injections of 20 to 25 mg/kg Ls-LAQ824 in nude mice with ErbB2 overexpressing BT-474 breast tumor xenografts significantly impairs tumor growth, and administration of ErbB2-targeted ILs-LAQ824 may further improve this antitumor activity. Studies of tumor-bearing mice 24 hours after single treatment indicate that: (a) >10% of injected liposomal LAQ824 is still circulating (whereas free LAQ824 is undetectable in the blood after 15 minutes); and (b) tumor uptake of Ls-LAQ824 and ILs-LAQ824 is >3% injected drug per gram of tumor, producing levels of acetylated tumor histones that are 5- to 10-fold increased over those following free LAQ824 or saline treatments and resulting in concordantly reduced levels of tumor ErbB2 mRNA. These preclinical results support the clinical evaluation of HDAC inhibitors against ErbB2-overexpressing malignancies, and further indicate that encapsulation into targeted and nontargeted liposomes substantially improves the in vivo pharmacokinetics, tumor uptake, and antitumor properties of hydroxamate-based HDAC inhibitors.

A mouse Chromosome 11 library generated from sorted chromosomes using linker-adapter polymerase chain reaction

We describe the generation of a mouse whole chromosome library using sequence-independent polymerase chain reaction (PCR) to amplify sequences contained in DNA extracted from flow sorted chromosomes. DNA in sorted chromosomes from a human x mouse hybrid cell line was digested with a frequent four-cutter restriction enzyme, Sau3AI, and the ends were ligated to an adapter oligonucleotide. The ligated DNA fragments were amplified using PCR primers homologous to the linker-adapter oligonucleotide. PCR-generated products were characterized by gel electrophoresis. The size of the amplified DNA ranged from 100 to more than 1,000 bp with a relatively high proportion of products at approximately 400 bp. Biotinylated PCR products used for FISH showed specific hybridization to murine metaphases and no hybridization to human lymphocyte and hamster metaphase cells. Banding analysis indicated that the probes were specific for mouse Chromosome 11. We anticipate that availability of painting probes for specific murine chromosomes will facilitate cytogenetic studies in the mouse.

Protein-protein interactions in hematology and phage display

Phage display, which exploits fundamental tools and principles of immune repertoire diversity, antigen-antibody interactions, and clonal and immunologic selection, is used increasingly to advance experimental and clinical hematology. Phage display is based on the ability of bacteriophage to present engineered proteins on their surface coat. Diverse libraries of proteins such as peptides, antibody fragments, and protein domains corresponding to gene fragments or cDNAs may be displayed. Interactions between phage-displayed proteins and target antigens can be identified rapidly and characterized using high throughput methodologies. Peptide and gene fragment libraries are particularly useful to characterize binding interactions between proteins, such as ligand-receptor interactions. This approach allows rapid generation of human antibodies, often against nonimmunogenic, conserved proteins. Phage antibodies against surface and intracellular antigens are used as reagents for flow cytometry, in vivo imaging, and therapeutic targeting. Phage-derived antibodies also facilitate analyses of the humoral antibody response. Finally, cellular delivery of phage-displayed peptides and gene fragments can be used to modulate functional pathways and molecules in vitro and in vivo. The combinatorial power of phage display enables identification of candidate epitopes without knowledge of the protein interaction, a priori. Overall, these capabilities provide a versatile, high-throughput approach to develop tools and reagents useful for a plethora of experimental hematology applications. This paper focuses on current and future applications of antibody and epitope phage display technology in hematology.

Molecular cytogenetics: Diagnosis and prognostic assessment

This review describes molecular cytogenetic techniques for detection and characterization of genetic aberrations associated with human disease. The techniques of fluorescence in situ hybridization, primed in situ labeling and comparative genome hybridization are described, as are probes for repeated sequences, whole chromosomes and specific loci. Also reviewed are applications of these technologies to pre- and neonatal diagnosis and to the characterization of human malignancies.