Flow cytometric DNA analysis as a diagnostic aid for cervical condyloma and cancer

Tsou, K. C.; Hong, Deokhwa; Varello, Michael; Giuntoli, Robert L.; Wheeler, James E.; Atkinson, Barbara; Mangan, Charles E.; Mikuta, John J.

doi:10.1002/1097-0142(19841101)54:9<1778::aid-cncr2820540905>3.0.co;2-s

Cited by 21 publications

(4 citation statements)

References 16 publications

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“…Histograms were plotted with a Hewlett Packard 7010B X-Y recorder (King of Prussia, PA). DNA histograms were corrected for peak deviations and CV (coefficient of variation) with a program written by the late Dr Walter Tsou (University of Pennsylvania-Presbyterian Medical Center) for an Apple computer (Tsou et a!, 1984).…”

Section: Methodsmentioning

confidence: 99%

Flow cytofluorometric analysis of granulosa cell proliferation in rats

Hirshfield¹,

Flickinger²,

Ben‐Rafael³

1988

Reproduction

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Immature rats were given PMSG followed by an ovulatory dose of hCG. Mature cyclic rats were given an ovulatory dose of hCG on the day of dioestrus, 24 h before the endogenous LH surge was expected to occur. Granulosa cells from individual follicles were subjected to flow cytometry at various times after hormone administration. Administration of PMSG to immature rats caused a marked, transient increase in 'cycling' granulosa cells (cells in S, G2, or M phases of the cell cycle). Administration of hCG to adult cyclic rats caused a slight, but significant, increase in 'cycling' granulosa cells. The results could be taken to indicate that hCG-induced luteinization is not, in fact, accompanied by cessation of cellular proliferation. However, the data lend themselves to alternative explanations. The observed phase shifts may be due to the lengthening of the duration of S and G2 phases, indicating an actual decrease in the rate of cell proliferation. Flow cytometry was found to be a rapid, inexpensive tool for the study of follicular growth in the rat, but, due to variation between follicles and the need for a minimal sample size of 2000 cells, its application is limited to large follicles.

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Section: Methodsmentioning

confidence: 99%

Flow cytofluorometric analysis of granulosa cell proliferation in rats

Hirshfield¹,

Flickinger²,

Ben‐Rafael³

1988

Reproduction

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“…The Percoll gradient-purified trophoblasts were fixed in 70% ethanol, stained with 4',6-diamidino-2-phenylindole, and analyzed with an impulse cytophotometer (ICP-22A, Ortho Instruments, Westwood, MA), as described by Tsou et al (18).…”

Section: Dna Content Analysismentioning

confidence: 99%

Purification, Characterization, andin vitroDifferentiation of Cytotrophoblasts from Human Term Placentae*

et al. 1986

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Highly purified functional cytotrophoblasts have been prepared from human term placentae by adding a Percoll gradient centrifugation step to a standard trypsin-DNase dispersion method. The isolated mononuclear trophoblasts averaged 10 microns in diameter, with occasional cells measuring up to 20-30 microns. Viability was greater than 90%. Transmission electron microscopy revealed that the cells had fine structural features typical of trophoblasts. In contrast to syncytial trophoblasts of intact term placentae, these cells did not stain for hCG, human placental lactogen, pregnancy-specific beta 1-glycoprotein or low mol wt cytokeratins by immunoperoxidase methods. Endothelial cells, fibroblasts, or macrophages did not contaminate the purified cytotrophoblasts, as evidenced by the lack of immunoperoxidase staining with antibodies against vimentin or alpha 1-antichymotrypsin. The cells produced progesterone (1 ng/10(6) cells . 4 h), and progesterone synthesis was stimulated up to 8-fold in the presence of 25-hydroxycholesterol (20 micrograms/ml). They also produced estrogens (1360 pg/10(6) cells . 4 h) when supplied with androstenedione (1 ng/ml) as a precursor. When placed in culture, the cytotrophoblasts consistently formed aggregates, which subsequently transformed into syncytia within 24-48 h after plating. Time lapse cinematography revealed that this process occurred by cell fusion. The presumptive syncytial groups were proven to be true syncytia by microinjection of fluorescently labeled alpha-actinin, which diffused completely throughout the syncytial cytoplasm within 30 min. Immunoperoxidase staining of cultured trophoblasts between 3.5 and 72 h after plating revealed a progressive increase in cytoplasmic pregnancy-specific beta 1-glycoprotein, hCG, and human placental lactogen concomitant with increasing numbers of aggregates and syncytia. At all time points examined, occasional single cells positive for these markers were identified. RIA of the spent culture media for hCG revealed a significant increase in secreted hCG, paralleling the increase in hCG-positive cells and syncytia identified by immunoperoxidase methods. We conclude that human cytotrophoblasts differentiate in culture and fuse to form functional syncytiotrophoblasts.

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“…A number of such procedures have been developed using both flow and microscope based image analysis systems. These include slit-scan nuclear cytoplasmic ratios (Wheeless et al, 1984), measurement of DNA content (Tsou et al, 1984;Fujii et al, 1984;Barrett et al, 1979;Tucker, 1979;Sprenger & Witte, 1979), DNA content plus total protein (Linden et al, 1979) and image analysis assaying DNA and chromatin 'texture' (Al & Ploem, 1979;Smeulders et al, 1979). These methods are based on either morphology or non-specific biochemical markers (DNA and total protein).…”

Section: Dna Analysesmentioning

confidence: 99%

Flow cytometric quantitation of DNA and c-myc oncoprotein in archival biopsies of uterine cervix neoplasia

Hendy-Ibbs¹,

Cox²,

Gi³

et al. 1987

Br J Cancer

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Summary The c-myc nuclear associated oncoprotein has been quantitated simultaneously with DNA in nuclei extracted from archival biopsies of uterine cervix neoplasia. The oncoprotein and DNA were measured fluorimetrically in a flow cytometer using a mouse monoclonal antibody (MYC I-6E10) and propidium iodide. Normal biopsies exhibited higher oncoprotein levels than carcinomas (P<0.00001). Furthermore, the maximum fluorescence signal in the normal tissue occurred at a lower antibody concentration compared with tumour tissue. There was no correlation between oncoprotein levels and histological grade, stage of disease, age of the patients or prognosis in the carcinomas. Aneuploidy, defined as a distinct second peak separate from the diploid distribution, was not a significant feature. The c-myc oncoprotein nuclear content does not appear to be a prognostic indicator in carcinoma of the cervix from the results of these studies but there is clearly diagnostic potential, particularly for automated analysis of cervical screening.Oncogcincs are associated with proliferation control. The csis gene encodes a subunit of platelet derived growth factor (Doolittle et al., 1983; Waterfield et al., 1983). v-erb B and cfms respectively encode the intracellular domain of epidermal growth factor receptor (Downward et al., 1984) and the transmembrane receptor for the macrophage colony stimulating factor, CSF 1, (Scherr et al., 1985). Expression of the c-myc gene is associated with the transition from a quiescent to a stimulated state (Kelly et al., 1983(Kelly et al., , 1984Makino et al., 1984;Greenberg & Ziff, 1984;Hann et al., 1985;Rabbitts et al., 1985).A series of mouse monoclonal antibodies which recognise the c-myc nuclear associated oncoprotein, p62c-mYc, (Evan & Hancock, 1985) have been developed . One of these antibodies has been used for histological localization of p62c-myc in both testicular cancer and in colonic neoplasia using immunocytochemical techniques. Quantitative methods have now been developed to assay nuclear associated p62cmvc in individual nuclei extracted from archival biopsies using flow cytometry . These methods have been used to analyse data in testicular cancer and colonic neoplasia (Watson et al., submitted). In this paper we examine the p62c-mYc nuclear content in normal and neoplastic cervical biopsies. Patients and methods PatientsA total of 127 patients attending the Weston Park Hospital Radiotherapy Department, Sheffield between 1971 and 1978 were included in the study. The only criteria of entry were that sufficient wax embedded punch biopsy material was available for the assay and that there were complete followup data. Sixty-four biopsies were from invasive carcinoma and 29 were from normal cervix. The remaining patients had cervical intraepithelial neoplasia (CIN) disease where 11, 9 and 14 were grades I, II and III respectively.Anti-p62c-mYc antibody Full details of the methods for production of the antip62c-mYc antibody are published elsewhere . Briefly, synthetic peptides were constructe...

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Flow cytometric DNA analysis as a diagnostic aid for cervical condyloma and cancer

Cited by 21 publications

References 16 publications

Flow cytofluorometric analysis of granulosa cell proliferation in rats

Flow cytofluorometric analysis of granulosa cell proliferation in rats

Purification, Characterization, andin vitroDifferentiation of Cytotrophoblasts from Human Term Placentae*

Flow cytometric quantitation of DNA and c-myc oncoprotein in archival biopsies of uterine cervix neoplasia

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