We have developed a procedure for simultaneous flow cytometric measurement of cellular DNA content and amount of BrdUrd incorporated into cellular DNA. Propidium iodide was used as a fluorescent probe for total cellular DNA and a monoclonal antibody against BrdUrd was used as a probe for BrdUrd incorporated into DNA. Fluorescein-labeled goat antimouse antibody was used to fluorescently label the bound antiBrdUrd probe. Bivariate DNA/BrdUrd distributions measured for Chinese hamster ovary cells labeled for 30 min with BrdUrd clearly show the G1-and G2M-phase cells to have low BrdUrd-linked fluorescence and the S-phase cells to have high BrdUrd-linked fluorescence. Cell cycle.traverse rates were estimated for Chinese hamster ovary cells from bivariate distributions measured-for samples taken periodically after pulse labeling with BrdUrdw Bivariate DNA/BrdUrd distributions were also applied in the analysis of the response of C3H murine bone marrow cells to treatment in vivo with l-I3-D-arabinofuranosylcytosine (araC). Bivariate distributions were measured for bone marrow cells taken from mice that were pulse labeled with BrdUrd at various times after treatment with araC. The resulting DNA/BrdUrd sequences show the kinetics of recovery from araC and allow discrimination of the araC sterilized cells.A broad range of biological and biomedical investigations depends on the ability to distinguish DNA synthesizing cells. Oncologists, for example, have devoted substantial effort to establishing correlations between the frequency of DNA synthesizing human tumor cells and the treatment prognosis (1). Effort has also been devoted to improvement of anticancer therapy with S-phase specific agents by treating when the experimentally determined frequency of tumor cells in S phase is maximal (2). In these studies, S-phase cells are usually assumed to be those that appear labeled in autoradiographs prepared immediately after pulse labeling with [3H]dThd or those with S-phase DNA content in DNA distributions measured flow cytometrically. Cytokineticists have relied heavily on measurements of the frequency of DNA synthesizing cells to determine the cell cycle traverse characteristics of normal and malignant cells. The classical "fraction of labeled mitosis" procedure (3), for example, depends on assessment of the frequency of mitotic cells that appear radioactively labeled in autoradiographs of samples taken periodically after labeling with [3H] We now report the development of a powerful, yet simple, flow cytometric procedure that promises to decrease or remove many of the limitations inherent in earlier methods for study of cell proliferation. Our procedure is based on the simultaneous flow cytometric measurement of cellular DNA content and amount of incorporated BrdUrd. For these measurements, propidium iodide (PrdI) is used as the probe for total DNA content and a monoclonal antibody against BrdUrd (8) Fig. 2 Upper. Ten thousand cells were sorted from each region directly into liquid scintillation vials. The radioactivi...

Current therapeutic approaches to cancer are designed to target molecules that contribute to malignant behavior but leave normal tissues intact. B 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 B 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 B 1 integrin signaling, resulting in malignant reversion. To investigate B 1 integrin as a therapeutic target, we modified the threedimensional 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 B 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 B 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 B 1 integrin is a promising therapeutic target, and that the threedimensional lrECM culture assay can be used to effectively distinguish malignant and normal tissue response to therapy.

One of the most frequent cytogenetic abnormalities in human leukemia and myelodysplasia is an interstitial deletion within chromosome 5q. A tumor suppressor gene has been hypothesized to lie in 5q31, the smallest commonly deleted region. IRF-1, a gene whose product manifests anti-oncogenic activity, was mapped to 5q31.1. IRF-1 lies between IL-5 and CDC25C and is centromeric to IL-3 and GM-CSF. Among these genes, only IRF-1 was consistently deleted at one or both alleles in 13 cases of leukemia or myelodysplasia with aberrations of 5q31. Inactivating rearrangements of one IRF-1 allele, accompanied by deletion of the second allele, were also identified in one case of acute leukemia. Thus, IRF-1 may be a critically deleted gene in human leukemia and myelodysplasia.

Challenges associated with the efficient and effective preparation of micro-and nano-scale (microand nano-gram) clinical specimens for proteomic applications include the unmitigated sample losses that occur during the processing steps. Herein we describe a simple "single-tube" preparation protocol appropriate for small proteomic samples using the organic co-solvent, trifluoroethanol (TFE) that circumvents the loss of sample by facilitating both protein extraction and protein denaturation without requiring a separate cleanup step. The performance of the TFE-based method was initially evaluated by comparisons to traditional detergent-based methods on relatively large scale sample processing using human breast cancer cells and mouse brain tissue. The results demonstrated that the TFE-based protocol provided comparable results to the traditional detergent-based protocols for larger, conventionally-sized proteomic samples (>100 μg protein content), based on both sample recovery and peptide/protein identifications. The effectiveness of this protocol for micro-and nano-scale sample processing was then evaluated for the extraction of proteins/peptides and shown effective for small mouse brain tissue samples (∼30 μg total protein content) and also for samples of ∼5 000 MCF-7 human breast cancer cells (∼500 ng total protein content), where the detergent-based methods were ineffective due to losses during cleanup and transfer steps.