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.

Separation of pancreatic islets by fluorescence-activated sorting.

To allow clinical pancreatic islet transplantation, the yield and purity of islets must be improved. Intravital staining of islets with neutral red is a specific, nontoxic technique for labeling islets of various species. Using neutral red-stained rat islets, we investigated the known fluorescence absorbance and emission spectra in comparison with unstained exocrine tissue and have shown that stimulation with light of wavelength between 500 and 560 nm produces detectable emission greater than 610 nm, which is absent from unstained exocrine tissue. The PARTEC cell sorter is an inexpensive alternative to currently available fluorescence-activated cell sorters and has a sorting mechanism based on a piezoelectric valve. We made extensive modifications to this machine to allow passage of particles up to 300 micron diam. Using rat pancreas stained intravitally with neutral red and dispersed by intraductal collagenase technique, we have shown that islets can be accurately identified in a high-speed flow system and sorted to a purity of greater than 90% islet tissue. The islets remain intact and viable as determined by supravital staining and isogeneic transplantation to the kidney capsule site. These studies prove the feasibility of separating intact islets by fluorescence-activated sorting.

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.

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.

Two-wavelength mercury arc lamp excitation for flow cytometric DNA-protein analyses.

Correlated DNA- and protein-determinations were performed using flow cytometry and DAPI-sulforhodamine 101 (SR101) staining. Two-wavelength UV-green excitation using a single HBO lamp was found to be superior to single wavelength UV excitation, since it resulted in a higher specificity in the SR101 measurements. The effects of overlapping in the emission spectra and of energy transfer from DAPI to SR101 were minimised. Different staining conditions and the specificity of SR101 staining were examined. No binding of SR101 to DNA and RNA could be detected. Good reproducibility was achieved in the protein measurements using human lymphocytes as an internal reference. Examples are given of the analysis of mixed cell populations with different cellular protein contents and different growth and metabolic activities. Correlated measurements of DNA and protein appear to be useful for further analysis of tumor cell populations.

The reproducibility of flow cytometric analyses in human tumors. Methodological aspects.

Various methodological aspects of flow cytometry were studied in material from endometrial carcinoma, ovarian tumors and bladder carcinoma. Measurements of identical samples on two different occasions gave an excellent correlation of the obtained DNA values, r = 0.997 and a good reproducibility of the S-phase rate, r = 0.87. In large tumors different DNA values were found in 5/36 cases when central and surface biopsies were compared (indicating tumor heterogeneity) which stresses the importance of multiple biopsies. The S-phase rate of the surface biopsies was generally higher. Comparing staining with ethidium bromide and DAPI, a good correspondence of ploidy determinations in human bladder tumors was found, provided that diploid cells from the normal tissue component were used as internal reference. When ethidium bromide staining was used, there was a good agreement between the values of tumor ploidy obtained by external standardization using lymphocytes and internal standardization using diploid tumor cells, respectively. In DAPI staining with lymphocytes as an external standard the tumor ploidy was systematically overestimated by about 10% due to suboptimal staining of lymphocytes after 3 hours. This difference decreased after 18 hours of staining. Determination of S-phase fraction showed a good correlation between DAPI and ethidium bromide stained bladder tumor samples (r = 0.86). Human lymphocytes as an external standard showed good reproducibility. In conclusion, flow cytometric measurements of the ploidy level and S-phase rate are highly reproducible provided that tumor heterogeneity is taken into account and proper preparation and standardization methods are used.

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.

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.

Radiation-induced diploid spermatids in mice.

Diploid elongated spermatids of mice were enriched by flow cytometry and cell sorting using a new type of sorter (Partec). The sorted abnormal spermatids were identified morphologically and by nuclear area integration. The radiation-induced increase in the frequency of diploid elongated spermatids was monitored with time following acute X-ray exposure of mice. Dose-response curves for acute 60Co-gamma and 14 MeV neutron irradiations yielded an RBE value of 4.3 for the doubling of the control level.

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.

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.

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.

Flow sorting of tumor cells for morphometric analysis, particularly of rare cells.

Using flow cytometric DNA measurement and sorting combined with morphometric light microscopy, different groups of cells were studied in a human melanoma pleural effusion, a human melanoma lymph node metastasis and a mouse tumor, as well as in normal reference tissues. Beside cells of the predominant tumor cell population, three types of rare tumor cells were studied after enrichment by sorting: a) giant cells from the greater than 8c region, comprising about 5% of the tumor cells, b) binucleated and multinucleated cells with unequal nuclear sizes within the same cell, found at frequencies of about 1.5%, and c) less than 2c cells which were derived from the so-called "debris"-region of the DNA histogram, found at frequencies of about 1 to 6%. All these rare cells were found only in the malignant tumors and not in the benign reference tissues. Morphometry showed that the increase in the cellular DNA content in the different fractions of tumor cells was combined with an increase in the cellular and nuclear sizes. However, the n/c-ratio was constant in the whole range of tumor cell fractions, including the fractions from the the less than 2c and the greater than 8c regions. The n/c-ratio of the less than 2c cells and giant cells differed from that of corresponding normal cells underlining their origin from the predominant tumor cell population. The possible linkage between the occurrence of the three rare cell types and genetic instability of tumors related to faulty nucleus and cell division is discussed.

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.

An improved Hedley method for preparation of paraffin-embedded tissues for flow cytometric analysis of ploidy and S-phase.

A modification of the Hedley-method for flow cytometric DNA analysis of paraffin-embedded tissues is presented. Dewaxed and dehydrated tissue from paraffin blocks was incubated with subtilisin Carlsberg (pronase, Sigma protease XXIV) and then stained directly without washing and centrifugation. The loss of material was minimized, which was advantageous, for example, for the analysis of core-biopsies, and all measured samples showed extremely low frequencies of clumped cell nuclei. This made is easier to detect polyploid nuclei and even rare nuclei of high ploidy could be identified. S-phase analyses were more precise, since the background originating from clumped debris particles was very low. The improved method was applied to the estimation of frequencies of high-polyploid nuclei found in various diploid, tetraploid, and aneuploid human myosarcomas of the uterus.

Improved method for release of cell nuclei from paraffin-embedded cell material of squamous cell carcinomas.

An improved method of releasing cell nuclei from paraffin-embedded highly keratinized squamous cell carcinomas by pretreatment with 85% formic acid-0.3% H2O2 followed by enzymatic treatment with subtilisin Carlsberg is described. After DAPI staining the resulting suspensions of cell nuclei were analyzed by DNA flow cytometry, in addition to microscopy. The total yield of released cell nuclei was improved and the proportion of tumor cell nuclei in the suspensions increased from 12% to 53% using this method compared to the conventional preparation technique. Cytoplasmic residue disappeared nearly completely. Thus, the finding of false aneuploid cell populations representing diploid cells with autofluorescent cytoplasm could be avoided. In addition, even small true aneuploid cell populations could be detected due to the increased proportion of released tumor cell nuclei and the lower proportion of background in the > 2C region.

DNA synthesis after combined treatment with cisplatin and 5-fluorouracil of a mouse ascites tumor growing in vivo.

We examined whether an increase in the salvage and/or the de novo synthesis of thymidine (TdR) can explain the elevated DNA synthesis rate found up to 15-20 h after combined treatment with cisplatin and 5-fluorouracil (5-FU), compared with single-drug regimen. The salvage and the de novo pathways of TdR in Bp8 mouse ascites tumor cells were reduced equally after the combined treatment and the single-drug treatments. The inhibition of the de novo pathway of TdR was confirmed by a reduced thymidylate synthase activity, as measured in cell extract. A marked imbalance of the deoxyribonucleotide triphosphates were found, in particular between the deoxypyrimidines. These imbalances were similar between the 5-FU single-drug treatment and combined treatment. We conclude that neither the extracellular TdR salvage nor the de novo synthesis of TdR explain the relatively elevated DNA synthesis rate after combined treatment. We suggest that the supra-additive effect of the combined treatment is due to an interaction between the elevated DNA synthesis, the imbalanced deoxyribonucleotides and the cisplatin-induced DNA cross-links, and possibly also due to a higher concentration of 5-FU incorporated into DNA.

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.

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.