Cell-cycle, protein content, and nuclear size in acute myeloid leukemia

Simultaneous analysis of DNA and cellular proteins provides information on cell proliferation and metabolism. Cellular protein content coupled with nuclear geometric parameters can be used to evaluate cellular maturation and differentiation. In this study, leucoblasts from 50 cases of adult acute myeloid leukemia were analyzed by flow cytometry, and semiautomatic morphometry was performed on bone marrow smears. Ethanol-fixed bone marrow blast cells were stained for DNA with propidium iodide (PI) and for proteins with fluorescein isothiocyanate (FITC). On the resulting FITC versus PI histograms we defined the cells with low protein content which are associated with a nonproliferating subpopulation (LPC fraction). Low protein content fraction and S-phase are correlated (p less than 0.01). The LPC fraction values are more dispersed than S-phase values and thus should indicate more clearly eventual differences between cellular populations. This hypothesis has been tested with the prognostic significance of cell-cycle variables: The LPC fraction was significantly higher in the complete remission group than in the other (p less than 0.01), while S-phase did not show any difference. The peak value of the protein content histograms is significantly lower in the granulocytic leukemias (M1, M2, M3) than in the leukemias with a monoblastic component (M4, M5). Furthermore, we showed that the differentiation and the maturation of the myeloid blast cells modify the nuclear size. The combination of these two parameters provides useful information for cytological classification.     

Response of 7,12-dimethylbenz(a)anthracene-induced rat mammary tumor cells to tamoxifen, in vitro

Primary cell cultures from a density-defined cell subpopulation of the DMBA-induced rat mammary tumors were exposed to tamoxifen during their log phase of growth. Growth inhibition and the ultrastructure of surviving cells were examined along with the influence of this antiestrogen on the secreted proteins as determined by pulse labeling with [35S]methionine and fluorography. Cell growth was remarkably inhibited at clinically achievable concentrations. However, ultrastructural changes in the surviving cells were minimal, the most noteworthy being the accumulation of myelin bodies. Protein secretion was affected in the defined subpopulation of several tumors by the reduced production of a high-molecular-weight protein. These tumors may represent a population of estrogen-sensitive tumors within the DMBA-induced mammary tumor model.     

Myelin basic protein-stimulated rosette-forming T cells in multiple sclerosis

A subpopulation of T lymphocytes sensitized to human myelin basic protein in peripheral blood of patients with multiple sclerosis, central nervous system (CNS) tumors, and cerebrovascular accidents was demonstrated by the antigen-stimulated, rosette-forming T cell assay. A significant increase in the percent of active rosette-forming T cells was detected after in vitro exposure of peripheral blood lymphocytes to human myelin basic protein but not to histones. In contrast, peripheral blood lymphocytes from healthy controls and from patients with benign and malignant breast diseases were unresponsive to stimulation by either antigen. These results demonstrate a functionally active T-lymphocyte subpopulation sensitized to myelin basic protein in patients with multiple sclerosis and in patients with certain other CNS diseases.     

Regulation of DNA synthesis in division-arrested mouse C127 cells permissive for bovine papillomavirus DNA amplification.

Spontaneous amplification of bovine papillomavirus type 1 DNA occurs following a prolonged period of serum starvation of wild-type virus-transformed C127 cell lines and is associated with abundant viral E2 protein synthesis and a concomitant induction of viral oncogene (E5 and E6) expression. We show here that a subpopulation of the permissive cells incorporate bromo-deoxyuridine under conditions of cell growth arrest (serum starvation), whereas DNA synthesis is suppressed in the resting population of nonpermissive cells. Flow cytometric measurements of the cellular DNA content of the permissive cell population indicated that it contained predominantly a 4n DNA content, suggesting that these cells were blocked in the G2 phase of the cell cycle. In keeping with the hypothesis that viral DNA amplification is associated with the induction of a cellular S phase, we observed a specific induction of expression of two cell proliferation-related cellular antigens (PCNA and Ki67) in a subpopulation of permissive cells. C127 cell lines transformed by an E5-minus bovine papillomavirus type 1 mutant, which was competent for autonomous plasmid replication in mitotic cells, were completely defective for the induction of DNA synthesis and mutant viral DNA amplification under conditions of serum starvation. Moreover, the E5 protein is shown by immunofluorescence analysis to be expressed at a high level specifically in the permissive cell population. These results imply a dual role for the viral E5 protein in the C127 model system, both as a transforming protein and as a factor required for the induction of viral DNA amplification in postmitotic cells. We suggest that E5 acts at an early step in the induction of this process in C127 cells and may be required to turn on host cell DNA synthesis as a prerequisite for viral DNA amplification.     

ABCG2-mediated DyeCycle Violet efflux defined side population in benign and malignant prostate

The efflux of Hoechst 33342 by ATP-binding cassette protein G2 (ABCG2) membrane pump allows reproducible identification of a subpopulation of cells by flow cytometric analysis termed the “side population” (SP). The SP identified by constitutive Hoechst efflux contains the stem/progenitor cell population from bone marrow and many solid organs, including prostate. DyeCycle Violet (DCV) is a cell membrane permeable, fluorescent vital dye that intercalates into DNA and is a substrate for ABCG2-mediated efflux. Therefore, DCV was evaluated in this study as a tool for identification of the SP from prostate cancer cell lines and from freshly harvested human prostate tissue. SPs that demonstrated ABCG2-mediated efflux of DCV were identified in the human prostate cancer cell lines CWR-R1, DU-145, and RWPE-1, but not in the BPH-1, LAPC-4, or PC-3 cell lines. Additionally, a SP was identified in enzymatically disaggregated prostate tumors from Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) human benign prostate tissue, and human prostate cancer tissue. The causal role of ABCG2-mediated efflux of DCV in the identification of the SP was confirmed by loss of the SP by incubation with the specific inhibitor of ABCG2, Fumitremorgin C. Expression of ABCG2 in the SP cells was confirmed by qRT-PCR and immunofluorescence analysis. Consequently, DCV represents an important new tool for isolation of viable candidate stem cells/cancer stem cells as a SP from cultured prostate cell lines, and prostate tissue specimens, without the requirement for instrumentation with ultra-violet excitation capability and minimizing the risk of damage to DNA in the sorted population.     

A cytophotometric and flow cytofluorometric approach to the differentiation of T lymphocytes in the thymus.

By cytophotometric and flow cytofluorometric DNA and protein determinations two main proliferating subpopulations of thymus lymphocytes with a different percentage of cells in the S phase could be distinguished. One subpopulation had a very low protein content, was cortisone sensitive and located in the cortex. Cells with comparable low protein contents were not found amongst lymphocytes of the peripheral blood. The other lymphocyte subpopulation had a higher protein content, was cortisone resistant and situated in the cortex around a group of epithelial cells and in the medulla. The protein content of these thymus lymphocytes appeared to be comparable to that of the peripheral blood lymphocytes. On the basis of the protein content per cell, it is possible to identify and isolate the more often described major subpopulation of cortisone sensitive thymus lymphocytes remaining and dying in the thymus, and the minor cortisone resistant subpopulation of thymus lymphocytes which is the source of the peripheral T lymphocyte.     

A cytophotometric and flow cytofluorometric approach to the differentiation of T lymphocytes in the thymus

Summary By cytophotometric and flow cytofluorometric DNA and protein determinations two main proliferating subpopulations of thymus lymphocytes with a different percentage of cells in the S phase could be distinguished. One subpopulation had a very low protein content, was cortisone sensitive and located in the cortex. Cells with comparable low protein contents were not found amongst lymphocytes of the peripheral blood. The other lymphocyte subpopulation had a higher protein content, was cortisone resistant and situated in the cortex around a group of epithelial cells and in the medulla. The protein content of these thymus lymphocytes appeared to be comparable to that of the peripheral blood lymphocytes. On the basis of the protein content per cell, it is possible to identify and isolate the more often described major subpopulation of cortisone sensitive thymus lymphocytes remaining and dying in the thymus, and the minor cortisone resistant subpopulation of thymus lymphocytes which is the source of the peripheral T lymphocyte.     

Germ cell nucleosomes contain remodeled core protein complex

Electrophoretic analysis of the nucleosomal histones from MN1 and MN2 subpopulations of the seminiferous tubules in gels containing either 6.25 or 2.5 M urea revealed the presence of testis specific histone H2S, H1 and protein ‘A’ in addition to the somatic histones in the core protein complex. Size analysis indicated the presence of a 150–160 bp DNA segment in the MNI subpopulation, whereas, an approx 180 bp DNA fragment was present in the MN2 subpopulation of both liver and tubule nucleosomes. These data suggest an extensive remodeling of the nucleosomal core protein complex during mammalian spermatogenesis.     

Dead cell discrimination with 7-amino-actinomycin D in combination with dual color immunofluorescence in single laser flow cytometry.

Identification of nonviable cells in immunofluorescently stained cell populations is essential for obtaining accurate data. Fluorescent non-vital DNA dyes, particularly propidium iodide (PI), have been used routinely in flow cytometry for discrimination of dead cells from viable cells on the basis of fluorescence. We describe here the use of an alternative DNA dye, 7-amino-actinomycin D (7-AAD), which can replace PI for the exclusion of nonviable cells. As an example, we present in this paper the utilization of 7-AAD on various leukemic cell lines for dead cell exclusion whenever the viable cell population could not be discriminated reliably from nonviable cells on the light scatter histogram; 7-AAD is suitable for dead cell discrimination in lengthy experiments because it is efficiently excluded by intact cells and has a high DNA binding constant. In addition, the dye is valuable in combination with phycoerythrin (PE)-fluorescence dual-color flow cytometry on a single argon laser instrument, since its emission in the far red can easily be separated from the emission of PE; 7-AAD was used on fluoresceinisothiocyanate (FITC) and PE surface-labeled human thymocytes for characterization of the dying subpopulation of cells which is undergoing programmed cell death. In this heterogeneous cell preparation, the spectral properties of the dye permitted the classification of viable and nonviable cell subpopulations by multiparameter analysis.     

How damaged is the biologically active subpopulation of transfected DNA?

Relatively little is known about the damage suffered by transfected DNA molecules during their journey from outside the cell into the nucleus. To follow selectively the minor subpopulation that completes this journey, we devised a genetic approach using simian virus 40 DNA transfected with DEAE-dextran. We investigated this active subpopulation in three ways: (i) by assaying reciprocal pairs of mutant linear dimers which differed only in the arrangement of two mutant genomes; (ii) by assaying a series of wild-type oligomers which ranged from 1.1 to 2.0 simian virus 40 genomes in length; and (iii) by assaying linear monomers of simian virus 40 which were cleaved within a nonessential region to leave either sticky, blunt, or mismatched ends. We conclude from these studies that transfected DNA molecules in the active subpopulation are moderately damaged by fragmentation and modification of ends. As a whole, the active subpopulation suffers about one break per 5 to 15 kilobases, and about 15 to 20% of the molecules have one or both ends modified. Our analysis of fragmentation is consistent with the random introduction of double-strand breaks, whose cause and exact nature are unknown. Our analysis of end modification indicated that the most prevalent form of damage involved deletion or addition of less than 25 base pairs. In addition we demonstrated directly that the efficiencies of joining sticky, blunt, or mismatched ends are identical, verifying the apparent ability of cells to join nearly any two DNA ends and suggesting that the efficiency of joining approaches 100%. The design of these experiments ensured that the detected damage preceded viral replication and thus should be common to all DNAs transfected with DEAE-dextran and not specific for viral DNA. These measurements of damage within transfected DNA have important consequences for studies of homologous and nonhomologous recombination in somatic cells as is discussed.     

Cytometry of breast carcinoma: significance of plo?dy balance and proliferation index

Image cytometry of DNA distribution in fine needle biopsies of breast carcinomas at first diagnosis was performed to see if there were significant differences in DNA histograms between patients having very different outcome but same tumor histological typing and similar therapy. Two groups of patients were considered retrospectively: the first (20 patients) with survival time shorter than 5 years and the second (20 patients) with survival time longer than 10 years. Seven benign tumors were used as controls. Ten plo?dy classes were defined. The frequencies of cells in those classes were used as independent features in a supervised multivariate analysis. The advantages of this approach was pointed out with respect to the four-type classification of Auer. The scattering of DNA histograms within the feature space showed that a subgroup of patients with poor prognosis was clearly separated from a subgroup of patients with good prognosis but both long survival patients and short survival patients were scattered in between. In order to replace the multivariate classification of histograms by a simpler approach, two parameters were computed which explained most of the scattering in the feature space: the plo?dy balance (difference between the percentages of euploid and aneuplo?d cells) and the proliferation index (percentage of cells between peaks). The scattergram of patients according to these parameters showed again that some DNA distributions were specific for either good or bad prognosis. But the separation was uncertain for seven short-survival patients and six long-survival patients. For six patients, the DNA distributions were very similar between long and short survival times. Those patients thus could not be separated even by means of discriminant analysis. The main conclusion of this study was that, for a significant number of patients, the objective multivariate classification of tumors DNA profiles is of little assistance to the pathologist who has to give a prognosis for the one patient under consideration.     

DNA ploidy in colorectal cancer, heterogeneity within and between tumors and relation to survival

Flow cytometry was used to study the incidence of aneuploidy and to determine the significance of multiple sampling from colorectal tumors. DNA ploidy pattern has been proposed as a supplementary prognostic marker, but discrepancies in findings are major. DNA clonal heterogeneity, defined as two or more DNA aneuploid stemlines in the same tumor, is well established. However, most studies have been based on only one biopsy from each tumor. In our study multiple biopsies were taken from 163 patients (88 males and 75 females) electively operated for colorectal cancer. Tumor cells were harvested by fine needle aspiration from fresh frozen biopsies sampled at different sites of each tumor. DNA aneuploidy was detected in tumors from 145 patients (89%), and 18 patients (11%) had a solitary DNA diploid cell population. In a 79 month follow-up period 105 patients had died. Statistical analysis showed that distinction between diploidy and aneuploidy did not predict survival. However, grouping subpopulations into DNA diploid plus near diploid (DNA index (DI) 0. 97-1.15), DNA aneuploid with all aneuploid subpopulations in the interval 1.15-2.06, and DNA aneuploid with subpopulations with DI < 0.97 and/or DI > 2.06, showed a significant difference in survival in a Cox multivariate analysis including Dukes' stage P = 0.049 comparing the second group to the first and P = 0.01 comparing the third group to the first. In 21 (13%) patients only one subpopulation was found, 57 (35%) had two, 44 (27%) had three, and 41 (25%) had four or more different subpopulations. The association of DNA ploidy to survival is shown to be dependent on the number of biopsies analysed.     

Stage-specific alterations of DNA methyltransferase expression, DNA hypermethylation, and DNA hypomethylation during prostate cancer progression in the transgenic adenocarcinoma of mouse prostate model

We analyzed DNA methyltransferase (Dnmt) protein expression and DNA methylation patterns during four progressive stages of prostate cancer in the transgenic adenocarcinoma of mouse prostate (TRAMP) model, including prostatic intraepithelial neoplasia, well-differentiated tumors, early poorly differentiated tumors, and late poorly differentiated tumors. Dnmt1, Dnmt3a, and Dnmt3b protein expression were increased in all stages; however, after normalization to cyclin A to account for cell cycle regulation, Dnmt proteins remained overexpressed in prostatic intraepithelial neoplasia and well-differentiated tumors, but not in poorly differentiated tumors. Restriction landmark genomic scanning analysis of locus-specific methylation revealed a high incidence of hypermethylation only in poorly differentiated (early and late) tumors. Several genes identified by restriction landmark genomic scanning showed hypermethylation of downstream regions correlating with mRNA overexpression, including p16INK4a, p19ARF, and Cacna1a. Parallel gene expression and DNA methylation analyses suggests that gene overexpression precedes downstream hypermethylation during prostate tumor progression. In contrast to gene hypermethylation, genomic DNA hypomethylation, including hypomethylation of repetitive elements and loss of genomic 5-methyldeoxycytidine, occurred in both early and late stages of prostate cancer. DNA hypermethylation and DNA hypomethylation did not correlate in TRAMP, and Dnmt protein expression did not correlate with either variable, with the exception of a borderline significant association between Dnmt1 expression and DNA hypermethylation. In summary, our data reveal the relative timing of and relationship between key alterations of the DNA methylation pathway occurring during prostate tumor progression in an in vivo model system.     

Flow cytometric analysis of DNA content and keratins by using CK7, CK8, CK18, CK19, and KL1 monoclonal antibodies in benign and malignant human breast tumors.

We have used a double-labelling flow cytometry analysis of keratin (CK) and DNA in breast cancer. Five monoclonal anti-keratin antibodies were tested: KL1 recognizing Mr 55,000-57,000 keratins, and "anti-glandular epithelia," LE41, RGE-53, and LP2K specific for CK n. 7, 8, 18, and 19 of Moll's classification, respectively. Flow cytometric (DNA-CK) analysis was performed on 10 benign and 19 malignant human breast tumors. All the benign tumors were diploid and 63% of the malignant tumors were aneuploid. This technique permits the analysis of DNA in the epithelial fraction alone. In aneuploid tumors, gating the DNA-keratin-positive population allowed accurate DNA analysis without interference due to debris background and non-epithelial cells. Moreover, double-labelling using the CK19 antibody gave a better identification of near-diploid tumors. An enhancement of keratin expression in malignant tumors was observed with CK 19 (P less than 0.001), KL1 (P less than 0.01), CK 8 (P less than 0.05), and CK18 (n.s.) compared to benign tumors. The comparison of keratin expression in aneuploid and diploid malignant tumors revealed reduced CK8, CK18, and CK19 in the former.     

Cytokinetic properties of asynchronous and cytosine arabinoside perturbed murine tumors measured by simultaneous bromodeoxyuridine/DNA analyses

This paper describes the determination of cytokinetic properties of asynchronous and cytosine arabinoside- (Ara-C) treated KHT tumors growing in vivo using the bromodeoxyuridine (BrdUrd)/DNA analysis technique. The cytokinetic properties of asynchronously growing tumors were estimated by computer analysis of sequential BrdUrd/DNA distributions measured at 2- to 3-h intervals after administration of a single i.p. injection of BrdUrd. The cytokinetic properties of the Ara-C-treated tumors were estimated by computer analysis of BrdUrd/DNA distributions measured at 2- to 3-h intervals after Ara-C treatment. BrdUrd was injected 30 min prior to tumor harvest. The cytokinetic properties of the cells rendered nonclonogenic by Ara-C were followed in BrdUrd/DNA distributions measured at 2- to 3-h intervals after Ara-C treatment of tumors that were labeled with BrdUrd 30 min prior to Ara C injection. The G1-, S-, and G2M-phase durations were estimated to be 7.6, 10.9, and 2.0 h prior to Ara-C; decreasing to 1.2, 4.1, and 1.4 after Ara-C. The growth fraction was estimated to be 0.8 prior to Ara-C. Complete recruitment of the normally noncycling subpopulation was observed after Ara-C treatment. Ara-C-killed cells were removed from the tumor within 24 h following Ara-C injection. These cytokinetic properties were similar to those reported in other studies.     

Overexpression of sigma1 receptor and its positive associations with pathologic TNM classification in esophageal squamous cell carcinoma

Sigma1 receptor (sigma1R), a significant protein, has been found to be frequently upregulated in human tumor cells and tissues. It has been demonstrated that sigma1R is involved in proliferation and adhesion of cancer cells. However, the significance of sigma1R expression in esophageal squamous cell carcinoma (ESCC) remains unclear. In this article, by a series of methods, the authors examined the expression of sigma1R protein in ESCC cell lines and tissues. Flow cytometry indicated intense staining of sigma1R in ESCC cells. Immunocytochemistry staining demonstrated that sigma1R was mainly distributed in cytoplasm and nucleus in ESCC cell lines. Western blotting was performed to characterize the relative expression of sigma1R in different ESCC cell lines. Moreover, different levels of sigma1R were presented from normal epithelium to carcinoma by immunohistochemistry analysis, which demonstrated that sigma1R was highly expressed in tumors. Association analysis showed significant correlations between total sigma1R protein levels and pathologic TNM (pTNM) classification of tumors (r=0.216, p=0.011). Furthermore, the sigma1R in the nucleus was significantly correlated with pTNM classification and lymph node metastasis (r=0.263, p=0.002, and r=0.269, p=0.002, respectively). These data indicated that sigma1R may serve as a potential predictive factor for pTNM classification and tumor development in ESCC.     

Flow cytometric DNA analysis of the human cervix affected by human papillomavirus and/or intraepithelial neoplasia

The relative DNA contents of 164 cellular samples from 59 patients affected by the viral cytopathic effects (VCE) of human papillomavirus (HPV) infection and/or by cervical intraepithelial neoplasia (CIN) and 12 cellular samples from 12 normal donors were analyzed by flow cytometry (FCM) with the aim of correlating the cytometric measurements with the morphologic and etiologic parameters. The unselected group of 59 patients was found to be characterized by statistically significant differences in average ages in the VCE and CIN (31.4 years) and CIN only (44.8 years) subgroups. Of the pathologic samples, 32 (54%) exhibited at least one cell subpopulation with an abnormal DNA content; in all but 2 of those cases, a diploid cell subpopulation was also present. The results indicate a relationship between the FCM ploidy and the morphologic classification, as shown by the increase in the occurrence of subpopulations with abnormal DNA contents from VCE only (38%) to VCE + CIN 1 (57%), to VCE + CIN 2/3 (70%). These results suggest that cytometric parameters, in association with the determination of the HPV types and in parallel to the colpocytohistopathologic criteria, can contribute to a more accurate characterization of cervical lesions in diagnostic and prognostic terms.     

A comparison between flow cytometric ploidy investigation and chromosome analysis of 32 human colorectal tumors

The correlation between flow cytometric ploidy investigation and classic chromosome analysis was studied in 32 human colorectal tumors. Flow cytometry was performed by nuclei isolation and DNA staining with ethidium bromide. Chromosome analysis was done after incubation with colcemid. In 12 cases, chromosome identification was possible by grouping according to the Denver system or by Q-banding. Generally, the measured DNA content corresponded well with the content expected from chromosome analysis, giving an average difference of 4%. In nine tumors, the measured DNA content was 4-18% higher than expected. Some of these discrepancies could be due to difficulties in identifying the corresponding cell populations in heterogeneous tumors. However, in general the number of cell populations and their quantitative representation by the two methods were statistically well correlated. The results indicate that flow cytometric ploidy investigation of colorectal tumors with the present technique is a reliable method, but also that a combination of both techniques may yield additional information about tumor cytogenetics.