Familiar and novel reproductive endocrine disruptors: xenoestrogens, dioxins and nanoparticles.

Environmental contaminants are known to exert endocrine-disrupting effects on the reproductive axis of animals. Many of these molecules can affect steroid biosynthesis or estrogen-receptor signaling by behaving as estrogen-like molecules ("xenoestrogens"), or by exerting estrogenmodulatory effects. Exposure to some compounds has been correlated with the skewing of sex ratios in aquatic species, feminization and demasculinization of male animals, declines in human sperm counts, and overall diminution in fertility of birds, fish, and mammals. We herein devote space to several classes of endocrine-disrupting compounds (EDCs), including estrogenic substances such as bisphenol A (BPA), molecules that can behave at times anti-estrogenically while activating the aromatic hydrocarbon receptor (AHR), such as dioxins (a known human carcinogen), and novel, ubiquitous molecules such as nanoparticles, particularly gold nanoparticles (GNPs), that appear to alter the sexsteroid biosynthetic pathway.

Environmental toxicants and effects on female reproductive function.

One of the most toxic substances known to humans, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin), is also highly pervasive in the environment. It is created naturally in volcanic eruptions and forest fires, and anthropogenically in waste incineration, chlorination processes and certain plastics manufacture. From reports of large industrial and other accidents, or from experimental studies, dioxin exposure has been correlated in animal models and/or humans with chloracne of the skin, organ cancers, hepatotoxicity, gonadal and immune changes, pulmonary and other diseases such as diabetes, skewing of the sex ratio, and infertility. We have demonstrated that the aromatic hydrocarbon receptor (AHR) that binds dioxin in tissues is localized in zebrafish, rat and rhesus monkey (Macaca mulatta) ovaries and in rat and human luteinizing granulosa cells (GC) (among other tissues), that labeled dioxin is specifically localized to granulosa cells of the ovarian follicle as observed by autoradiography, and that incubations of GC or ovarian fragments with environmentally relevant concentrations (fM to nM) of dioxin inhibit estradiol secretion significantly. Our experiments show that in human, non-human primate, rat, trout, and zebrafish ovarian tissues, dioxin inhibits estrogen synthesis at some level of the steroid biosynthetic pathway, most likely by inhibiting transcription of mRNAs for or activity of side-chain cleavage (Cyp11a1 gene) and/or aromatase (Cyp19a1 gene) enzymes, or conceivably other steroidogenic enzymes/factors. Such an untoward effect on estrogen synthesis in females exposed to dioxin environmentally may predispose them to defects in aspects of their fertility.

Dose and time dependent apoptotic response in a human melanoma cell line exposed to accelerated boron ions at four different LET.

The aim was to investigate and compare the influence of linear energy transfer (LET), dose and time on the induction of apoptosis in a human melanoma cell line exposed to accelerated light boron ((10)B) ions and photons. Cells were exposed in vitro to doses up to 6 Gy accelerated boron ions (40, 80, 125 and 160 eV nm(-1)) and up to 12 Gy photons (0.2 eV nm(-1)). The induction of apoptosis was measured up to 9 days after irradiation using morphological characterization of apoptotic cells and bodies. In parallel, measurements of cell-cycle distribution, monitored by DNA flow cytometry, and cell survival based on the clonogenic cell survival assay, were performed. In addition, the induction and repair of DNA double-strand breaks (DSB), using pulsed-field gel electrophoresis (PFGE) were studied. Accelerated boron ions induced a significant increase in apoptosis as compared with photons at all time points studied. At 1-5 h the percentage of radiation-induced apoptotic cells increased with both dose and LET. At the later time points (24-216 h) the apoptotic response was more complex and did not increase in a strictly LET-dependent manner. The early premitotic apoptotic cells disappeared at 24 h following exposure to the highest LET (160 eV nm(-1)). A postmitotic apoptotic response was seen after release of the dose-, time- and LET-dependent G2/M accumulations. The loss of clonogenic ability was dose- and LET-dependent and the fraction of un-rejoined DSB increased with increasing LET. Despite the LET-dependent clonogenic cell killing, it was not possible to measure quantitatively a LET-dependent apoptotic response. This was due to the different time course of appearance and disappearance of apoptotic cells.

Noninvasive estimation of tumour viability in a xenograft model of human neuroblastoma with proton magnetic resonance spectroscopy (1H MRS).

The aim of the study was to evaluate proton magnetic resonance spectroscopy ((1)H MRS) for noninvasive biological characterisation of neuroblastoma xenografts in vivo. For designing the experiments, human neuroblastoma xenografts growing subcutaneously in nude rats were analysed in vivo with (1)H MRS and magnetic resonance imaging at 4.7 T. The effects of spontaneous tumour growth and antiangiogenesis treatment, respectively, on spectral characteristics were evaluated. The spectroscopic findings were compared to tumour morphology, proliferation and viable tumour tissue fraction. The results showed that signals from choline (Cho)-containing compounds and mobile lipids (MLs) dominated the spectra. The individual ML/Cho ratios for both treated and untreated tumours were positively correlated with tumour volume (P<0.05). There was an inverse correlation between the ML/Cho ratio and the viable tumour fraction (r=-0.86, P<0.001). Higher ML/Cho ratios concomitant with pronounced histological changes were seen in spectra from tumours treated with the antiangiogenic drug TNP-470, compared to untreated control tumours (P<0.05). In conclusion, the ML/Cho ratio obtained in vivo by (1)H MRS enabled accurate assessment of the viable tumour fraction in a human neuroblastoma xenograft model. (1)H MRS also revealed early metabolic effects of antiangiogenesis treatment. (1)H MRS could prove useful as a tool to monitor experimental therapy in preclinical models of neuroblastoma, and possibly also in children.

Expression of CTLA-4 by human monocytes.

Cytotoxic T lymphocyte-associated molecule-4 (CTLA-4) is a receptor present on T cells that plays a critical role in the downregulation of antigen-activated immune responses. CTLA-4 interacts with the ligands CD80 and CD86 on antigen-presenting cells (APC), and also directs the assembly of inhibitory signalling complexes that lead to quiescence or anergy. In this study, we show that human monocytes constitutively express CTLA-4. About 3% of monocytes expressed CTLA-4 on the cell surface, whereas the intracellular expression was higher and present in about 20% of the monocytes. The sequences of the cDNAs from human monocytes were identical to the sequences of CTLA-4 from T cells. Expression of CTLA-4 was also confirmed in the activated myelomonocytic cell lines U937 and THP-1. Monocytes, but not T cells, activated by interferon (IFN)-gamma also secreted soluble CTLA-4 in vitro. The CTLA-4 expression was upregulated upon treatment with phorbol 12-myristate 13-acetate (PMA) and IFN-gamma. This increased expression could be partially abolished by staurosporine, an inhibitor of protein kinase C (PKC). Ligation of CTLA-4 in the monocyte-like cell-line U937 with antibodies against CTLA-4 partially inhibited the proliferation of cells and the upregulation of cell-surface markers CD86, CD54, HLA-DR and HLA-DQ induced by IFN-gamma and Staphylococcus aureus, Cowan I strain (SAC). Ligation of CTLA-4 suppressed the PMA-stimulated activation of transcription activator protein 1 (AP-1) and nuclear factor (NF)-kappaB in the U937 cell line, indicating the involvement of an inhibitory signal transduction. These data provide the first evidence that CTLA-4 is constitutively expressed by monocytes and thus might be important for the regulation of immune mechanisms associated with monocytes.

The MEK1 inhibitor PD98059 sensitizes C8161 melanoma cells to cisplatin-induced apoptosis.

The regulation of apoptosis is believed to be dependent on the balance of the activities of different intracellular signalling systems. Activation of the SAPK/JNK pathway is implied in pro-apoptotic signalling, while activation of the MEK1/ERK pathway may have a viability-promoting effect. We show here that treatment with the MEK1 inhibitor PD98059 sensitizes the human melanoma cell line C8161 to cisplatin-induced apoptosis. In these cells, cisplatin at 40 microM did not elicit significant cell death, whereas massive cell death was seen when cells were pretreated for 20 h with 40 microM PD98059 before the addition of cisplatin. Concomitant addition of PD98059 and cisplatin did not have any sensitizing effect, and PD98059 on its own did not induce apoptosis. However, in three other human melanoma cell lines PD98059 did not potentiate cisplatin-induced apoptosis. Instead, in one of these cell lines (AA), PD98059 protected against cisplatin-induced cytotoxicity. We conclude that blocking of the MEK1/ERK pathway may, in some instances, potentiate the cytotoxic effect of cisplatin on human melanoma cell lines, whereas in other instances it may have a protective effect. Thus it cannot be regarded as a general approach to sensitizing melanoma cells to drug-induced apoptosis.

Proliferation and apoptosis in the evolution of endemic and acquired immunodeficiency syndrome-related Kaposi's sarcoma.

Kaposi's sarcoma (KS) is a multifocal lesion that occurs predominantly in the skin, most frequently in people infected with HIV-1, and that evolves through early stages (patch and plaque) to a tumor-like late stage (nodular). Both, endemic African (EKS) and AIDS-associated (AKS) KS expressed human herpesvirus 8 (HHV-8) as shown by PCR. By immunohistochemistry the expression of cellular Bcl-2 and c-myc was confined in early stages of both EKS and AKS to relatively few endothelial cells (EC) whereas in nodular KS most of spindle cells (SC) strongly expressed both genes. CD40 was usually strongly expressed in SC at all KS stages as well as in EC of non-involved tissue whereas CD40L (CD154) was not demonstrable. Fas (CD95) was moderately to weakly expressed by SC whereas p53 and Waf-1 were found in less than 5% of the SC. In both AKS and EKS at nodular stage almost no apoptotic SC were detected. In most AKS and EKS low levels of cell proliferation were seen but AKS showed consistently higher values compared to EKS. All clinical types and stages of KS showed a diploid cellular DNA content by flow cytometric analysis of microselected lesions. Thus, we conclude that KS during evolution represents diploid, probably reactive, cell proliferation, which progressively increases the expression of strong cellular and also viral (HHV-8) antiapoptotic factors.

Combined analysis of DNA ploidy, proliferation, and apoptosis in paraffin-embedded cell material by flow cytometry.

A flow cytometric assay was developed for correlated measurement of DNA content and apoptotic DNA strand breaks in cell nuclei of formalin-fixed, paraffin-embedded tissues. The assay allows a combined analysis of cell ploidy, proliferation, and apoptosis in sections of fixed paraffin-embedded archival or fresh tissue/cell specimens. It is based on (a) proteolytic release of cell nuclei from deparaffinized and rehydrated 90-microm thick sections of the fixed embedded specimen, (b) the inactivation of the protease, (c) FITC-labeling of DNA strand breaks by the terminal deoxynucleotidyl transferase (TdT)-mediated FITC-dUTP nick end-labeling (TUNEL) reaction, and (d) DNA staining with 4'6-diamidino-2-phenyleindole. The fluorescence was recorded with a double-beam flow cytometer equipped with a mercury arc lamp and an argon ion laser. Cytograms obtained with this assay correlated closely with those produced using nonembedded material from the same specimen. Furthermore, a significant correlation was found between flow cytometric analysis of apoptosis in cell nuclei released from paraffin blocks and conventional evaluation of TUNEL on (corresponding) sections (p < 0.001). Since necrotic cells can stain positively by TUNEL, the possibility to microscopically select nonnecrotic tumor regions for flow cytometric analysis is an important advantage of the assay.

Reliability of DNA cytometric S-phase analysis in surgical biopsies: assessment of systematic and sampling errors and comparison between results obtained by image and flow cytometry.

Three major parameters in DNA histograms that contribute to the reliability of S-phase analysis were evaluated. These parameters are (1) the extent of background in relation to the amount of S-phase cells (and the validity of its subtraction), (2) the size of the "free" S-phase range (S(free)), and (3) the sampling error of cell counting. Tests in histograms obtained from surgical biopsies by flow cytometry (FCM) showed that the background subtraction is reliable if the found S-phase fraction is higher than the fraction of background events in the histogram range of the cell population. The size of S(free) was determined in computer-generated test histograms as a function of variables such as the coefficient of variation (CV) and the DNA index (DI). To calculate the sampling error of cell counting above background and in S(free), a model was developed that was validated by experimental data. This error can serve as an indicator of the uncertainty in S-phase analysis. The poor correlation found between %S values measured by image cytometry (ICM) and FCM in surgical biopsies was assigned to high uncertainty by low cell numbers in ICM histograms. A method is proposed to estimate quantitatively the reliability of S-phase analysis that can facilitate the interpretation of results.

Increased apoptosis and increased clonogenic survival of 12V-H-ras transformed rat fibroblasts in response to cisplatin.

Mutationally activated Ras is involved in tumor progression and likely also in drug resistance. Using survival, viability and apoptosis assays, we have here compared the cisplatin sensitivities of FR3T3 rat fibroblasts and a 12V-H-ras transformed subline (Ras2:3). Around 24 h after cisplatin treatment Ras2:3 cells showed higher apoptosis levels and lower viability than FR3T3. This increased sensitivity correlated with weaker cisplatin-induced activation of Jun N-terminal kinase (JNK). In contrast to apoptosis assays, colony formation assays showed that Ras2:3 were more resistant to cisplatin than were FR3T3. This was partly due to the increased cisplatin sensitivity of FR3T3 seeded at low densities, as required in colony formation assays. In addition, Ras2:3 cisplatin survivors had a higher relative proliferative capacity. Cell cycle analyses showed that FR3T3 cells initially responded with a dose-dependent G2 arrest, while Ras2:3 accumulated in S-phase. Experiments with an anti-apoptotic mutant of MEKK1 suggested that the apoptotic response of Ras2:3 cells is not specific to the S-phase fraction. In summary, the cisplatin response of ras-transformed fibroblasts is distinct from that of parental cells, in that they show increased apoptosis, a different cell cycle response and increased post-treatment proliferative capacity. The results illustrate the need to carefully consider methods and protocols for in vitro studies on chemotherapy sensitivity.

Molecular mechanisms underlying interferon-alpha-induced G0/G1 arrest: CKI-mediated regulation of G1 Cdk-complexes and activation of pocket proteins.

One prominent effect of IFNs is their cell growth-inhibitory activity. The mechanism behind this inhibition of proliferation is still not fully understood. In this study, the effect of IFN-alpha treatment on cell cycle progression has been analysed in three lymphoid cell lines, Daudi, U-266 and H9. Examination of the growth-arrested cell populations shows that Daudi cells accumulate in a G0-like state, whereas U-266 cells arrest later in G1. H9 cells are completely resistant to IFN-alpha's cell growth-inhibitory effects. The G0/G1-phase arrest is preceded by a rapid induction of the cyclin-dependent kinase inhibitors (CKIs), p21 and p15. In parallel, the activities of the G1 Cdks are significantly reduced. In addition to p21/p15 induction, IFN-alpha regulates the expression of another CKI, p27, presumably by a post-transcriptional mechanism. In the G1 Cdk-complexes, there is first an increased binding of p21 and p15 to their respective kinases. At longer exposure times, when Cdk-bound p15 and p21 decline, p27 starts to accumulate. Furthermore, we found that IFN-alpha not only suppresses the phosphorylation of pRb, but also alters the phosphorylation and expression of the other pocket proteins p130 and p107. These data suggest that induction of p21/p15 is involved in the primary IFN-alpha response inhibiting G1 Cdk activity, whereas increased p27 expression is part of a second set of events which keep these Cdks in their inactive form. Moreover, elevated levels of p27 correlated with a dissociation of cyclin E/Cdk2-p130 or p107 complexes to yield cyclin E/Cdk2-p27 complexes. In resistant H9 cells, which possess a homozygous deletion of the p15/p16 genes and lack p21 protein expression, IFN-alpha causes no detectable changes in p27 expression and, furthermore, no effects are observed on either pocket proteins in this cell line. Taken together, these data suggest that the early decline in G1 Cdk activity, subsequent changes in phosphorylation of pocket proteins, and G1/G0 arrest following IFN-alpha treatment, is not primarily due to loss of the G1 kinase components, but result from the inhibitory action of CKIs on these complexes.

Proliferation and apoptosis-related gene expression in experimental acquired immunodeficiency syndrome-related simian lymphoma.

Lymphomas in 10 cynomolgus monkeys infected with a simian immunodeficiency virus (SIVsm) were studied with regard to proliferative activity and apoptosis-related gene expression. All were diffuse large-cell lymphomas, showed mono or oligoclonality and a 9/10 diploid cellular DNA content. Expression of a simian homologue to Epstein-Barr virus (HVMF-1) was shown in nine cases. The lymphomas showed moderate to high proliferative activity by Ki67 immunostaining and DNA flow cytometry, and a low number of apoptotic cells detected by TdT-mediated dUTP nick-end labeling (TUNEL). Immunohistochemistry showed abundant tumor infiltrating TIA-1(+) cytotoxic lymphocytes (CTL) and macrophages. Bcl-2, Mcl-1, and also Bax and Bak, but not p53 were demonstrable in the tumor cells by immunostaining. Our findings suggest a causal relationship between HVMF-1 infection and a low apoptotic index of the lymphomas due to the expression of Bcl-2. The apparent inefficient function of tumor-infiltrating CTL could be due to inactivation of CTL and/or resistance of the lymphoma cells to CTL effects. The tumors showed immunoreactivity for CD18, CD29, and CD49d, but not for CD11a, mimicking the phenotype of human Epstein-Barr virus (EBV)-related lymphomas. In summary, our observations indicate a high similarity between this simian model of acquired immunodeficiency syndrome (AIDS)-related lymphomas (ARL) and human ARL and other immunosuppression-related lymphomas.

Comparison of routine flow cytometric DNA analysis of fresh tissues in two laboratories: effects of differences in preparation methods and background models of cell cycle calculation.

Routine flow cytometric DNA analysis was compared in two laboratories by using matched fresh-frozen breast cancer and soft tissue sarcoma biopsy specimens. Laboratory I applied the Vindelöv preparation method and an exponential background subtraction algorithm in the cell cycle calculation. Laboratory II used the Formalin-protease preparation technique and the sliced-nuclei background model. The results of the ploidy analysis showed good agreement between the two laboratories; however, the results of the cell cycle analysis showed considerable systematic differences between labs. Laboratory I obtained significantly lower values of S-phase fraction and higher values of G2-phase fraction than laboratory II. To explain these discrepancies, the effects of differences in the preparation methods and background subtraction algorithms were studied. The Vindelöv preparation method yielded higher debris and aggregation levels than the Formalin-protease technique and tended to give higher %S and %G2 values. When the two background models were used in the same histograms, the exponential background model tended to give %S values distinctly lower than and %G2 values almost identical to those obtained with the sliced-nuclei algorithm. The sum of these effects accounts for the observed inter-laboratory discrepancies. Different from the sliced-nuclei fit, the exponential background fit often did not accommodate to the original data in the <2c histogram region and resulted in a considerable inter-operator variability of %S calculation in histograms with <5% S. When aggregate correction was added to the sliced-nuclei algorithm, the differences between %S values in histograms from the two laboratories almost disappeared.

Comparison between radiation-induced cell cycle delay in lymphocytes and radiotherapy response in head and neck cancer.

A study was made evaluating the use of radiation-induced cell cycle delay in lymphocytes to predict tumour response to radiotherapy. Peripheral blood lymphocytes were isolated from whole blood from 49 patients with head and neck cancer before treatment with radiotherapy and from 25 healthy donors. The clinical response to radiotherapy was assessed at 0-2 months after treatment. The level of radiation-induced cell cycle delay was measured using flow cytometry after mitogen stimulation of lymphocytes. The analysis of ten normal donors gave no significant difference in variability between the intra-assay and the intra-donor samples. However, the cell cycle data for lymphocytes from these healthy donors showed significant inter-individual differences in G2 phase accumulation. Patients showing no response to radiotherapy had a high level of S-phase cells compared with partial (P < 0.001) and complete responders (P = 0.016). An inverse relationship was found when analysing the fraction of cells in G2 (P = 0.009 and 0.034 respectively). In general, healthy donors had similar cell cycle kinetics compared with the non-responders. In conclusion, the result indicates that radiation-induced cell cycle delay in lymphocytes is inversely correlated with tumour response to radiotherapy in head and neck cancer patients. However, the value of the present test for predicting individual tumour response is limited, because of assay variability and overlap between groups.

Characterisation of the G1/S cell cycle checkpoint defect in lung carcinoma cells with different intrinsic radiosensitivities.

Cell cycle perturbations in three lung carcinoma cell lines (U-1285,U1906 and U-1810) with different intrinsic radiosensitivities (SF2 U-1285 = 0.25, SF2 U-1906 = 0.45, SF2 U-1810 = 0.88) were investigated following x-irradiation. Cell cycle flow calculations showed that the G1-->S-phase transit was accelerated in irradiated compared with untreated U-1285 cells, up to 24 hours postirradiation. In U-1810 cells and U-1906 cells the postirradiation G1-->S transit decreased compared with controls. All three cell lines showed no postirradiation induction of p53 and p21CIP1 proteins. Cyclin E was overexpressed and cyclin E-dependent kinase activity was substantially induced by irradiation in U-1285 cells compared with U-1906 and U1810 cells while p27KIP1 was detected at the highest intensity in U-1810 cells and lowest in U-1285 cells. We hypothesise that the accelerated postirradiation G1-->S transit in U-1285 cells is associated with induction of cyclin E-dependent kinase activity and may account for increased radiosensitivity in these cells.

Induction of apoptosis and inhibition of cell growth are independent responses to interferon-alpha in hematopoietic cell lines.

IFNs are capable of modulating a variety of cellular responses, including cell growth and apoptosis. The prospective connections between these two biological responses are not fully understood, and the molecular mechanisms underlying the effects of IFNs on these processes are not completely defined. We have investigated the relationship between IFN-alpha-induced apoptosis and cell cycle arrest in three hematopoietic cell lines, Daudi, U-266, and H9. It was found that IFN-alpha was a rapid and potent inducer of apoptosis in H9 and U-266 cells, whereas IFN-alpha-induced cell cycle arrest in Daudi cells is not associated with the onset of apoptosis. In H9 cells, apoptosis occurs without a preceding cell cycle block, whereas in U-266 cells, apoptosis occurs subsequent to G1 arrest. Cell cycle arrest per se, induced by serum starvation or treatment with aphidicolin, had only minor effects on the viability of these cell lines and did not abrogate the apoptosis-inducing capacity of IFN-alpha. Additionally, IFN-alpha-induced apoptosis occurred in cells from all cell cycle phases. Thus, we conclude that IFN-alpha-induced apoptosis seems to occur independent of cell growth inhibition. There were no changes in Bcl-2 or Bax protein levels that could account for the apoptosis-inducing effects of IFN-alpha in these cell lines. Moreover, examination of p53 status suggests that IFN-alpha-induced apoptosis in the U-266 and H9 cell lines occurs through a p53-independent pathway.

Non-tumorigenic SV40T immortalized human buccal epithelial cells show aneuploidy and genetic instability.

Cytogenetic and DNA flow cytometric analysis was carried out on four SV40T-transfected human buccal epithelial cells lines. One of these was immortalized and showed a nontumorigenic phenotype when tested in athymic nude mice. DNA flow cytometric ploidy values correlated well with cytogenetic ploidy values as calculated from chromosome length or DNA content, whereas the chromosome counts correlated poorly with the flow cytometric results. Gross ploidy changes were seen at early passages, while the immortalized cell line had a stabilized DNA content in the near diploid range. However, this cell line showed ongoing random chromosomal changes with the appearance of new marker chromosomes balancing chromosome losses. The chromosome losses were mainly found in the groups 12-16 and 18-23 and the gains in the group 1-6. This reflects, together with the stabilization of the DNA content, a nonrandom component in the overall random chromosomal changes. In conclusion, aneuploidy and genetic instability found in the immortalized cell line were not linked to malignant growth in nude mice.

Fluorescence image cytometry for measurement of nuclear DNA content in surgical pathology.

A study was made on various methodological aspects of fluorescence image cytometry (FICM) for measurement of nuclear DNA content by using CCD cameras attached to an epifluorescence microscope. Cell nuclei of paraffin-embedded specimens from mouse tissues and human prostate carcinomas were isolated and stained with 4'-6-diamidino-2-phenylindole (DAPI). We found that fluorescence fading, lamp stability, and the homogeneity of the illumination can easily be controlled. A camera with a signal-to-noise ratio of 53 dB gave a slightly more precise measurement than did a 46-dB camera. The linearity of the analysis results was very good. The coefficient of variation of mouse kidney standard cells in the DNA histograms was about 5% and 7.4% in histograms of prostate carcinoma biopsies. Stained cell nuclei can be stored for long periods at -20 degrees C without impairment of quality. Comparative measurements of ploidy by FICM and flow cytometry confirmed the accuracy of the FICM analyses. Thus, FICM appears to be an easy method for quantifying the DNA content of visually inspected cell nuclei in surgical pathology.

Cell cycle regulation of immunoglobulin class switch recombination and germ-line transcription: potential role of Ets family members.

Previous studies have indicated that transcription of germ-line (GL) CH genes is necessary to obtain immunoglobulin (Ig) class switching. We report here a correlation between proliferation, switching and GL transcripts. Smu-S gamma 1 switch recombination in lipopolysaccharide (LPS) + interleukin-4 (IL-4)-activated mouse B cells was assayed by a digestion-circularization polymerase chain reaction. Switching to gamma 1 is reduced upon inhibition of DNA synthesis with hydroxy-urea (HU) or aphidicholin (AC). Incubation of activated B cells with HU severely reduces steady-state levels of GL gamma 1 and epsilon RNA. By utilizing elutriation to synchronize B cell blasts in different phases of the cell cycle, it was found that GL gamma 1 transcripts are mainly expressed in G1 and S phases, but not in G0. Using the electrophoretic mobility shift assay, we characterized two major LPS-induced complexes, which bind to the GL gamma 1 promoter and are expressed at levels which correlate with the amount of LPS-induced DNA synthesis. Furthermore, the intensity of the complexes is reduced when cells are arrested with the DNA synthesis inhibitors HU or AC. Elutriation experiments revealed that the complexes are expressed in G1 and S, but not in G0. They bind to an Ets consensus element near the major initiation sites used in proliferating cells. The possible implications of these findings for Ig isotype switching are discussed.

New epi-fluorescence optical system for independent analysis of two different fluorochromes in microscopy.

A new epi-fluorescence optical system is described that uses splitting of the primary excitation and emission light beams, independent modification of the separated beams, and their reunification. The optical system was constructed for analysis of two different fluorochromes, e.g., DAPI and TRITC. Modifications in the separated beams comprise: (1) isolation of specific wavelengths (365 nm, 546 nm, 435-500 nm, and 590-750 nm), (2) wavelength switching without image displacement and blur by means of a light chopper alternating between ultraviolet-excitation/blue-detection and green-excitation/red-detection at frequencies of up to 140 Hz for observation by eye without image flicker, and (3) separate positioning of lenses for compensation of chromatic aberrations. This system demonstrates a good transmission of the chosen wavelengths. A high specificity of double fluorescence analysis with minimal effects of spectral overlap was obtained with good temporal resolution. It has been shown that it is feasable to obtain separate chromatic compensations for the use of a microscope objective in spectral regions outside the range for which the objective is corrected.