Cytometry of chromatin bound Mcm6 and PCNA identifies two states in G1 that are separated functionally by the G1 restriction point.

BACKGROUND: Cytometric measurements of DNA content and chromatin-bound Mcm2 have demonstrated bimodal patterns of expression in G1. These patterns, the replication licensing function of Mcm proteins, and a correlation between Mcm loading and cell cycle commitment for cells re-entering the cell cycle, led us to test the idea that cells expressing a defined high level of chromatin-bound Mcm6 in G1 are committed--i.e., past the G1 restriction point. We developed a cell-based assay for tightly-bound PCNA (PCNA*) and Mcm6 (Mcm6*), DNA content, and a mitotic marker to clearly define G1, S, G2, and M phases of the cell cycle. hTERT-BJ1, hTERT-RPE-1, and Molt4 cells were extracted with Triton X-100 followed by methanol fixation, stained with antibodies and DAPI, then measured by cytometry. RESULTS: Bivariate analysis of cytometric data demonstrated complex patterns with distinct clustering for all combinations of the 4 variables. In G1, cells clustered in two groups characterized by low and high Mcm6* expression. Serum starvation and release experiments showed that residence in the high group was in late G1, just prior to S phase. Kinetic experiments, employing serum withdrawal, and stathmokinetic analysis with aphidicolin, mimosine or nocodazole demonstrated that cells with high levels of Mcm6* cycled with the committed phases of the cell cycle (S, G2, and M). CONCLUSIONS: A multivariate assay for Mcm6*, PCNA*, DNA content, and a mitotic marker provides analysis capable of estimating the fraction of pre and post-restriction point G1 cells and supports the idea that there are at least two states in G1 defined by levels of chromatin bound Mcm proteins.

A new biomarker for mitotic cells.

Many epitopes are phosphorylated during mitosis. These epitopes are useful biomarkers for mitotic cells. The most commonly used are MPM-2 and serine 10 of histone H3. Here we investigated the use of an antibody generated against a phospho peptide matching residues 774-788 of the human retinoblastoma protein 1 (Rb) to detect mitotic cells. Human cell lines were stained with DNA dyes and antibodies reactive with epitopes defined by antibody MPM-2, phospho-S10-histone-H3, and the phospho-serine peptide, TRPPTLSPIPHIPRC (phospho-S780-Rb). Immunoreactivity and DNA content were measured by flow and image cytometry. Correlation and pattern recognition analyses were performed on list mode data. Western blots and immunoprecipitation were used to investigate the number of peptides reactive with phospho-S780-Rb and the relationship between reactivity with this antibody and MPM-2. Costaining for bromodeoxyuridine (BrdU) was used to determine acid resistance of the phospho-S780-Rb epitope. Cell cycle related phospho-S780-Rb immunofluorescence correlated strongly with that of MPM-2. Laser scanning cytometry showed that phospho-S780-Rb immunofluorescence is expressed at high levels on all stages of mitotic cells. Western blotting and immunoprecipitation showed that the epitope is expressed on several peptides including Rb protein. Costaining of BrdU showed that the epitope is stable to acid. Kinetic experiments showed utility in complex cell cycle analysis aimed at measuring cell cycle transition state timing. The phospho-S780-Rb epitope is a robust marker of mitosis that allows cytometric detection of mitotic cells beginning with chromatin condensation and ending after cytokinesis. Costaining of cells with DNA dyes allows discrimination and counting of mitotic cells and post-cytokinetic ("newborn") cells. To facilitate use without confusion about specificity, we suggest the trivial name, pS780 for this mitotic epitope.

Phase I and correlative study of combination bryostatin 1 and vincristine in relapsed B-cell malignancies.

PURPOSE: Bryostatin 1 activates protein kinase C (PKC) with short-term exposure and results in depletion of PKC with prolonged exposure. Preclinical in vitro and in vivo studies demonstrate synergistic activity and increased tumor apoptosis in B-cell malignancies when a prolonged infusion of bryostatin 1 is followed by vincristine. EXPERIMENTAL DESIGN: We embarked on a Phase I trial of bryostatin 1 as a 24-h continuous infusion followed by bolus vincristine in patients with refractory B-cell malignancies other than acute leukemias. Twenty-four evaluable patients were enrolled. RESULTS: The dose-limiting toxicity was myalgia. The MTD and recommended Phase II dose of bryostatin 1 was 50 microg/m2/24 h followed by vincristine 1.4 mg/m2 (maximum total dose of 2 mg) repeated every 2 weeks. Significant antitumor activity was observed in this relapsed population, including patients who had failed high-dose chemotherapy. This included 5 durable complete and partial responses and 5 patients with stable disease lasting > or =6 months (range, 6-48+ months). Median time to response was 8 months. Correlative studies demonstrated a progressive increase in serum interleukin-6 with bryostatin 1 infusion followed by an additional increase after vincristine. Flow cytometry for detection of apoptosis in B and T cells showed an initial decrease in apoptotic frequency in CD5+ cells within 6 h of bryostatin 1 infusion compatible with its known increase in PKC activity in the majority of patients followed by a return to baseline or overall increase in apoptotic frequency after completion of infusion. All (5 of 5) patients who had an overall increase in apoptotic frequency in CD5+ cells achieved either a clinical response or prolonged stable disease. Four of these 5 patients did not have the initial decrement in apoptosis at 6 h. CONCLUSIONS: Given the lack of myelosuppression and early evidence of clinical efficacy, additional exploration of this regimen in non-Hodgkin's lymphoma and multiple myeloma is warranted.

Cell density related gene expression: SV40 large T antigen levels in immortalized astrocyte lines.

BACKGROUND: Gene expression is affected by population density. Cell density is a potent negative regulator of cell cycle time during exponential growth. Here, we asked whether SV40 large T antigen (Tag) levels, driven by two different promoters, changed in a predictable and regular manner during exponential growth in clonal astrocyte cell lines, immortalized and dependent on Tag. RESULTS: Expression and cell cycle phase fractions were measured and correlated using flow cytometry. T antigen levels did not change or increased during exponential growth as a function of the G1 fraction and increasing cell density when Tag was transcribed from the Moloney Murine Leukemia virus (MoMuLV) long terminal repeat (LTR). When an Rb-binding mutant T antigen transcribed from the LTR was tested, levels decreased. When transcribed from the herpes thymidine kinase promoter, Tag levels decreased. The directions of change and the rates of change in Tag expression were unrelated to the average T antigen levels (i.e., the expression potential). CONCLUSIONS: These data show that Tag expression potential in these lines varies depending on the vector and clonal variation, but that the observed level depends on cell density and cell cycle transit time. The hypothetical terms, expression at zero cell density and expression at minimum G1 phase fraction, were introduced to simplify measures of expression potential.

Growth, immortalization, and differentiation potential of normal adult human proximal tubule cells.

Human proximal tubule epithelial cell lines are potentially useful models to elucidate the complex cellular and molecular details of water and electrolyte homeostasis in the kidney. Samples of normal adult human kidney tissue were obtained from surgical specimens, and S1 segments of proximal convoluted tubules were microdissected, placed on collagen-coated culture plate inserts, and cocultured with lethally irradiated 3T3 fibroblasts. Primary cultures of proximal tubule epithelial cells were infected with a replication-defective retroviral construct encoding either wild-type or temperature-sensitive simian virus 40 large T-antigen. Cells forming electrically resistive monolayers were selected and expanded in culture. Three cell lines (HPCT-03-ts, HPCT-05-wt, and HPCT-06-wt) were characterized for proximal tubule phenotype by electron microscopy, electrophysiology, immunofluorescence, Southern hybridization, and reverse transcriptase-polymerase chain reaction. Each of the three formed polarized, resistive epithelial monolayers with apical microvilli, tight junctional complexes, numerous mitochondria, well-developed Golgi complexes, extensive endoplasmic reticulum, convolutions of the basolateral plasma membrane, and a primary cilium. Each exhibited succinate, phosphate, and Na,K- adenosine triphosphatase (ATPase) transport activity, as well as acidic dipeptide- and adenosine triphosphate-regulated mechanisms of ion transport. Transcripts for Na(+)-bicarbonate cotransporter, Na(+)-H(+) exchanger isoform 3, Na,K-ATPase, parathyroid hormone receptor, epidermal growth factor receptor, and vasopressin V2 receptor were identified. Furthermore, immunoreactive sodium phosphate cotransporter type II, vasopressin receptor V1a, and CLIC-1 (NCC27) were also identified. These well-differentiated, transport-competent cell lines demonstrated the growth, immortalization, and differentiation potential of normal, adult, human proximal tubule cells and consequently have wide applicability in cell biology and renal physiology.

Cell cycle-related cyclin b1 quantification.

BACKGROUND: To obtain non-relative measures of cell proteins, purified preparations of the same proteins are used as standards in Western blots. We have previously quantified SV40 large T antigen expressed over a several fold range in different cell lines and correlated the average number of molecules to average fluorescence obtained by cytometry and determined cell cycle phase related expression by calculation from multi-parametric cytometry data. Using a modified approach, we report quantification of endogenous cyclin B1 and generation of the cell cycle time related expression profile. METHODOLOGY: Recombinant cyclin B1 was purified from a baculovirus lysate using an antibody affinity column and concentrated. We created fixed cell preparations from nocodazole-treated (high cyclin B1) and serum starved (low cyclin B1) PC3 cells that were either lyophilized (for preservation) or solubilized. The lysates and purified cyclin B1 were subjected to Western blotting; the cell preparations were subjected to cytometry, and fluorescence was correlated to molecules. Three untreated cell lines (K562, HeLa, and RKO) were prepared for cytometry without lyophilization and also prepared for Western blotting. These were quantified by Western blotting and by cytometry using the standard cell preparations. RESULTS: The standard cell preparations had 1.5 x 10(5) to 2.5 x 10(6) molecules of cyclin B1 per cell on average (i.e., 16-fold range). The average coefficient of variation was 24%. Fluorescence varied 12-fold. The relationship between molecules/cell (Western blot) and immunofluorescence (cytometry) was linear (r(2) = 0.87). Average cyclin B1 levels for the three untreated cell lines determined by Western blotting and cytometry agreed within a factor of 2. The non-linear rise in cyclin B1 in S phase was quantified from correlated plots of cyclin B1 and DNA content. The peak levels achieved in G2 were similar despite differences in lineage, growth conditions, and rates of increase through the cell cycle (range: 1.6-2.2 x 10(6) molecules per cell). CONCLUSIONS: Net cyclin B1 expression begins in G1 in human somatic cells lines; increases non-linearly with variation in rates of accumulation, but peaks at similar peak values in different cell lines growing under different conditions. This suggests tight quantitative end point control.

Immunoreactivity of Stat5 phosphorylated on tyrosine as a cell-based measure of Bcr/Abl kinase activity.

BACKGROUND: Stat5(1) (Signal Transducer and Activator of Transcription 5) is normally phosphorylated and activated by Janus kinases. In cells transformed with BCR/ABL, Stat5 is constitutively activated by promiscuous phosphorylation. Cytometry of intracellular antigens can be used to evaluate cell treatments affecting gene expression, because it precisely provides the fraction of affected cells and the quantitative change in expression. Here, we asked whether we could measure a phosphorylated epitope on Stat5 by cytometry, and whether that measurement would respond to Bcr/Abl inhibition. METHODS: Chronic myelogenous leukemia (CML) cell lines or control Bcr/Abl-negative cells were treated or not with imatinib mesylate, fixed and permeabilized with formaldehyde followed by methanol; reacted with rabbit polyclonal and mouse monoclonal antibodies against an epitope including tyrosine 694 of Stat5a (pSTAT5); reacted with antibodies that mark mitotic cells; counterstained with secondary fluorescent antibodies and 4',6-diamidino-2-phenylindole (DAPI); and then subjected to flow cytometry. Western blotting was performed with pSTAT5 and Stat5 antibodies. RESULTS: Optimal fixation and staining parameters were established for pSTAT5 antibodies with K562 cells. These cells displayed high levels of immunoreactivity with pSTAT5 probes that could be inhibited uniformly with imatinib mesylate in a dose-response and time-dependent manner. The IC50 for downregulation of pSTAT5 immunoreactivity for K562 cells by cytometry was approximately 70 nM. The inhibition half-time was approximately 1 min. At micromolar doses this reactivity remained minimal for up to 7 days. Cultured cells also displayed a population of negative cells that increased under conditions related to cessation of cell growth (media nutrient depletion). This study also showed quantitatively that a rabbit polyclonal antibody that cross-reacted with an additional epitope could be used successfully as a measure of Bcr/Abl activity. CONCLUSION: We have developed a sensitive cytometric assay for Bcr/Abl kinase activity in human hematopoietic cell lines.