DNA methyltransferase levels in tumorigenic and nontumorigenic cells in culture

The levels of DNA methyltransferase in nuclei from 9 tumorigenic and 9 nontumorigenic cell lines were examined. In all but 2 cases, the extractable methyltransferase activity was 4-3000-fold higher in tumorigenic than in nontumorigenic cells. Tumorigenic and nontumorigenic cells from four species were grown in the presence of various concentrations (10(-8)-10(-6) M) of an inhibitor of the methylase enzyme, 5-aza-2'-deoxycytidine (5-aza-dCyd). The reduction of 5-methylcytosine content in newly replicated DNA in the presence of 5-aza-dCyd was used to determine the relative methylase activity in each cell line. In all 4 cases, tumorigenic cells required larger doses of drug to inhibit DNA methylation to the same extent as their nontumorigenic counterparts. The relative rates of incorporation of [3H]5-aza-dCyd were determined for each cell line, and tumorigenic cells were shown to incorporate equal or greater amounts of 5-aza-dCyd into DNA compared to nontumorigenic cells. These results showed that the differences in the inhibition of DNA methylation in response to 5-aza-dCyd were not due to differences in the ability of these cells to incorporate the drug. Thus, it was demonstrated by two independent methods that tumorigenic cells contained higher levels of methylating capacity than nontumorigenic cells. This overabundance of methyltransferase may alter DNA methylation patterns and affect phenotypic stability.     

FTIR spectroscopy demonstrates biochemical differences in mammalian cell cultures at different growth stages

We have observed differences in the infrared spectra of viable fibroblast cells depending on whether the cells were in the exponential (proliferating) or plateau (nonproliferating) phase of growth. The spectral changes were observed even after correcting for cell number and volume, ruling out these trivial explanations. Several of the changes occurred for both transformed and normal cell lines and were greater for the normal cell line. The biochemical basis of the spectral changes was estimated by fitting the cell spectra to a linear superposition of spectra for the major biochemical components of mammalian cells (DNA, RNA, protein, lipids, and glycogen). The ratios of RNA/lipid and protein/lipid increased when the cells were in the exponential phase compared to the plateau phase of growth. The fits of cell spectra to individual biochemical components also demonstrated that DNA is a relatively minor spectroscopic component as would be expected biochemically. Contrary to other reports in the literature, our data demonstrate that determining DNA content or structure using Fourier transform infrared spectroscopy data is difficult due to the relatively small amount of DNA and the overlap of DNA bands with the absorption bands of other biochemical components.     

Evaluation of mitochondrial function and metabolic reprogramming during tumor progression in a cell model of skin carcinogenesis

Metabolic reprogramming from mitochondrial aerobic respiration to aerobic glycolysis is a hallmark of cancer. However, whether it is caused by a dysfunction in the oxidative phosphorylation pathway is still under debate. In this work, we have analyzed the bioenergetic cellular (BEC) index and the relative cell ability to grow in the presence of either galactose or glucose as sources of sugar (Gal/Glu index) of a system formed by four epidermal cell lines with increasing tumorigenic potentials, ranging from nontumorigenic to highly malignant. We find that the BEC index gradually decreases whereas the Gal/Glu index increases with tumorigenicity, indicating that a progressive metabolic adaptation to aerobic glycolysis occurs in tumor cells associated with malignancy. Interestingly, this metabolic adaptation does not appear to be caused by damaged respiration, since the expression and activity of components of the respiratory chain complexes were unchanged in the cell lines. Moreover, the corresponding mitochondrial ATP synthetic abilities of the cell lines were found similar. The production of reactive oxygen species was also measured. A shift in ROS generation was found when compared nontumorigenic with tumorigenic cell lines, the latter exhibiting about threefold higher ROS levels than nontumorigenic cells. This result indicates that oxidative stress is an early event during tumor progression.     

In vitro toxicology evaluation of pharmaceuticals using Raman micro-spectroscopy

Raman micro-spectroscopy combined with multivariate analysis was employed to monitor real-time biochemical changes induced in living cells in vitro following exposure to a pharmaceutical. The cancer drug etoposide (topoisomerase II inhibitor) was used to induce double-strand DNA breaks in human type II pneumocyte-like cells (A549 cell-line). Raman spectra of A549 cells exposed to 100 microM etoposide were collected and classical least squares (CLS) analysis used to determine the relative concentrations of the main cellular components. It was found that the concentrations of DNA and RNA significantly (P < 0.05) decreased, whilst the concentration of lipids significantly (P < 0.05) increased with increasing etoposide exposure time as compared to control untreated A549 cells. The concentration of DNA decreased by 27.5 and 87.0% after 24 and 48 h exposure to etoposide respectively. Principal components analysis (PCA) successfully discriminated between treated and untreated cells, with the main variance between treatment groups attributed to changes in DNA and lipid. DNA fragmentation was confirmed by Western blot analysis of apoptosis regulator protein p53 and cell metabolic activity determined by MTT assay. The over-expression of p53 protein in the etoposide treated cells indicated a significant level of DNA fragmentation and apoptosis. MTT tests confirmed that cellular metabolic activity decreased following exposure to etoposide by 29.4 and 61.2% after 24 and 48 h, respectively. Raman micro-spectroscopy may find applications in the toxicology screening of other drugs, chemicals and new biomaterials, with a range of cell types.     

Identification of a human chromosome 11 gene which is differentially regulated in tumorigenic and nontumorigenic somatic cell hybrids of HeLa cells.

The tumorigenicity of HeLa cells in nude mice can be suppressed by the addition of a normal human chromosome 11 in somatic cell hybrids. We have attempted to identify specific genes involved in this phenomenon by transfecting a complementary DNA expression library into a tumorigenic HeLa-fibroblast hybrid. A cell line designated F2 was isolated which displayed morphological features of the nontumorigenic hybrids, demonstrated reduced tumorigenicity in nude mice, and showed an 85% reduction in alkaline phosphatase, a consistent marker of the tumorigenic phenotype in these cells. F2 contained a single exogenous complementary DNA, which was recovered by polymerase chain reaction and designated HTS1 because of its potential association with "HeLa tumor suppression." Northern blot studies suggested differential regulation of the HTS1 gene dependent on the tumorigenicity of the cell. In nontumorigenic hybrids, RNA species of 2.8, 3.1, and 4.6 kilobases were identified. In two tumorigenic hybrid lines, the 2.8-kilobase species was markedly reduced or absent. Similarly, three nontumorigenic human keratinocyte lines expressed all three RNA species, whereas several tumorigenic cervical carcinoma cell lines lacked the 2.8-kilobase species. Chromosome localization studies mapped the HTS1 gene to chromosome 11p15, a region of chromosome 11 that is believed to contain a tumor suppressor gene. These findings indicate that HTS1 represents a novel chromosome 11 gene which may be a target of the tumor suppressor gene active in this system.     

Non-invasive analysis of cell cycle dynamics in single living cells with Raman micro-spectroscopy

Raman micro-spectroscopy is a laser-based technique which enables rapid and non-invasive biochemical analysis of cells and tissues without the need for labels, markers or stains. Previous characterization of the mammalian cell cycle using Raman micro-spectroscopy involved the analysis of suspensions of viable cells and individual fixed and/or dried cells. Cell suspensions do not provide cell-specific information, and fixing/drying can introduce artefacts which distort Raman spectra, potentially obscuring both qualitative and quantitative analytical results. In this article, we present Raman spectral characterization of biochemical changes related to cell cycle dynamics within single living cells in vitro. Raman spectra of human osteosarcoma cells synchronized in G(0)/G(1), S, and G(2)/M phases of the cell cycle were obtained and multivariate statistics applied to analyze the changes in cell spectra as a function of cell cycle phase. Principal components analysis identified spectral differences between cells in different phases, indicating a decrease in relative cellular lipid contribution to Raman spectral signatures from G(0)/G(1) to G(2)/M, with a concurrent relative increase in signal from nucleic acids and proteins. Supervised linear discriminant analysis of spectra was used to classify cells according to cell cycle phase, and exhibited 97% discrimination between G(0)/G(1)-phase cells and G(2)/M-phase cells. The non-invasive analysis of live cell cycle dynamics with Raman micro-spectroscopy demonstrates the potential of this approach to monitoring biochemical cellular reactions and processes in live cells in the absence of fixatives or labels.     

Viral RNA Synthesis and Levels of DNA-Dependent RNA Polymerases During Replication of Adenovirus 2

The rates of RNA synthesis in cultured human KB cells infected by adenovirus 2 were estimated by measuring the endogenous RNA polymerase activities in isolated nuclei. The fungal toxin α-amanitin was used to determine the relative and absolute levels of RNA synthesis by RNA polymerases I, II, and III in nuclei isolated during the course of infection. Whereas the level of endogenous RNA polymerase I activity in nuclei from infected cells remained constant relative to the level in nuclei from mock-infected cells, the endogenous RNA polymerase II and III activities each increased about 10-fold. These increases in endogenous RNA polymerase activities were accompanied by concomitant increases in the rates of synthesis in isolated nuclei of viral mRNA precursor, which was monitored by hybridization to viral DNA, and of viral 5.5S RNA, which was quantitated by electrophoretic analysis on polyacrylamide gels. The cellular RNA polymerase levels were measured with exogenous templates after solubilization and chromatographic resolution of the enzymes on DEAE-Sephadex, using procedures in which no losses of activity were apparent. In contrast to the endogenous RNA polymerase activities in isolated nuclei, the cellular levels of the solubilized class I, II, and III RNA polymerases remained constant throughout the course of the infection. Furthermore, no differences were detected in the chromatographic properties of the RNA polymerases obtained from infected or control mock-infected cells. These observations suggest that the increases in endogenous RNA polymerase activities in isolated nuclei are not due to variations in the cellular concentrations of the enzymes. Instead, it is likely that the increased endogenous enzyme activities result from either the large amounts of viral DNA template available as a consequence of viral replication or from functional modifications of the RNA polymerases or from a combination of these effects.     

Viral RNA synthesis and levels of DNA-dependent RNA polymerases during replication of adenovirus 2.

The rates of RNA synthesis in cultured human KB cells infected by adenovirus 2 were estimated by measuring the endogenous RNA polymerase activities in isolated nuclei. The fungal toxin alpha-amanitin was used to determine the relative and absolute levels of RNA polymerases I, II, and III in nuclei isolated during the course of infection. Whereas the level of endogenous RNA polymerase I activity in nuclei from infected cells remained constant relative to the level in nuclei from mock-infected cells, the endogenous RNA polymerase II and III activities each increased about 10-fold. These increases in endogenous RNA polymerase activities were accompanied by concomitant increases in the rates of synthesis in isolated nuclei of viral mRNA precursor, which was quantitated by electrophoretic analysis on polyacrylamide gels. The cellular RNA polymerase levels were measured with exogenous templates after solubilization and chromatographic resolution of the enzymes on DEAE-Sephadex, using procedures in which no losses of activity were apparent. In contrast to the endogenous RNA polymerase activities in isolated nuclei, the cellular levels of the solubilized class I, II, and III RNA polymerases remained constant throughout the course of the infection. Furthermore, no differences were detected in the chromatographic properties of the RNA polymerases obtained from infected or control mock-infected cells. These observations suggest that the increases in endogenous RNA polymerase activities in isolated nuclei are not due to variations in the cellular concentrations of the enzymes. Instead, it is likely that the increased endogenous enzyme activities result from either the large amounts of viral DNA template available as a consequence of viral replication of from replication or from functional modifications of the RNA polymerases or from a combination of these effects.     

Functional evidence for a second tumor suppressor gene on human chromosome 17.

The development and progression of human tumors often involves inactivation of tumor suppressor gene function. Observations that specific chromosome deletions correlate with distinct groups of cancer suggest that some types of tumors may share common defective tumor suppressor genes. In support of this notion, our initial studies showed that four human carcinoma cell lines belong to the same complementation group for tumorigenic potential. In this investigation, we have extended these studies to six human soft tissue sarcoma cell lines. Our data showed that hybrid cells between a peripheral neuroepithelioma (PNET) cell line and normal human fibroblasts or HeLa cells were nontumorigenic. However, hybrid cells between the PNET cell line and five other soft tissue sarcoma cell lines remained highly tumorigenic, suggesting at least one common genetic defect in the control of tumorigenic potential in these cells. To determine the location of this common tumor suppressor gene, we examined biochemical and molecular polymorphic markers in matched pairs of tumorigenic and nontumorigenic hybrid cells between the PNET cell line and a normal human fibroblast. The data showed that loss of the fibroblast-derived chromosome 17 correlated with the conversion from nontumorigenic to tumorigenic cells. Transfer of two different chromosome 17s containing a mutant form of the p53 gene into the PNET cell line caused suppression of tumorigenic potential, implying the presence of a second tumor suppressor gene on chromosome 17.     

Behavior of HEp-2 cell line cells, their nuclei and nucleoli during cultivation and under the effect of Na-ds-RNA

The behavior of human larynx cancer cells (HEp-2) and of their nuclei and nucleoli during the cultivation without the influence of Na-ds-RNA and after its introduction into the medium was investigated by methods of cytomorphometry and cytophotometry. The density of monolayer (the number of cells on the area unit), percentage of two-nuclear cells, the number of nucleoli in the nuclei, mitotic coefficient, volume and total surface of nuclei and nucleoli have been measured. In addition, the mass of DNA in the nuclei and that of the total RNA and DNA in the nuclei and in each nucleolus was measured. Cells in the culture, not subjected to the influence of Na-ds-RNA, were weakly differentiated, kept active proliferation, and their population contained a small number of two-nuclear elements and a high share of multi-nuclear cell. During cultivation, these indices became even more pronounced, which is typical for the increase in cell malignancy. Under the influence of Na-ds-RNA, the proliferate activity decreases, the number of double-nuclear cells increases, while that of multi-nucleolar cell decreases; also, the share of cells with one- and two-nucleolar nuclei increases. The authors conclude that Na-ds-RNA may have antineoplastic activities, clearly evidenced from its influence on the culture of transformed HEp-2 cells.     

Desmoplakin expression and distribution in cultured rat bladder epithelial cells of varying tumorigenic potential

The expression and distribution of the desmosomal plaque proteins, desmoplakins (DPs) I and II, were studied in nontumorigenic (RBE-8) and a series of tumorigenic (AY34, R-4909, SS-24B, RBTCC-8, and 804G) rat bladder epithelial cell lines. These cell lines ranged from slow-growing papillary transitional cells (AY34) to rapidly metastatic carcinoma cells (RBTCC-8). DPs I and II were shown by immunoblotting and Northern analysis to be present in nontumorigenic RBE-8 cells as well as in all of the tumorigenic cell lines, albeit in differing amounts. Immunofluorescence microscopy revealed striking differences in DP distribution, corresponding in general with increases in tumorigenic potential. Whereas DPs of normal RBE-8 cells and less tumorigenic AY34 cells were localized predominantly at cell interfaces, the more tumorigenic lines exhibited a high proportion of DP in the form of cytoplasmic dots, a distribution reminiscent of that seen in epithelial cells maintained in low levels of extracellular calcium. In 804G cells, which represented the most extreme example of this phenomenon, the majority of DPs were organized as cytoplasmic dots. Electron microscopy revealed intermediate filament (IF)-associated spots in the cytoplasm as well as an elaborate array of IF-associated plaques at the cell-substratum interface. The IF-associated spots in the cytoplasm reacted with anti-DP antibody in immunogold labeling experiments while those at the cell-substratum did not react. In more dense cultures of 804G cells, certain cells stratified and expressed increased amounts of DP followed by the induction of new keratins including those of the skin type. Decreasing extracellular calcium resulted in a rearrangement of DP in each cell line; staining at cell-cell interfaces disappeared and was replaced with a pattern of cytoplasmic dots. These results demonstrate a possible relationship between desmosome assembly and/or maintenance and tumorigenic potential.     

Raman Spectroscopy Analysis of the Biochemical Characteristics of Molecules Associated with the Malignant Transformation of Gastric Mucosa

Objective

The purpose of this study was to comparatively analyze the signature Raman spectra of genomic DNA, nuclei, and tissue of normal gastric mucosa and gastric cancer and to investigate the biochemical transformation of molecules associated with gastric mucosa malignancy.

Method

Genomic DNA, nuclei, and tissue from normal gastric mucosa and gastric cancer were analyzed by Raman spectroscopy.

Results

1) The Raman spectrum of gastric cancer genomic DNA showed that two peaks appeared, one at approximately 1090 cm^-1 with a higher intensity than the peak at 1050 cm^-1 in the spectrum. Characteristic peaks appeared at 950 cm^-1, 1010 cm^-1, and 1100-1600 cm^-1. 2) Using a hematoxylin and eosin (H&E)-stained section, the intensity of the characteristic peak of nucleic acids at 1085 cm^-1 was increased and shifted to 1088 cm^-1 in cancer cells. The relative intensity of the characteristic peaks of nucleoproteins at 755 cm^-1 and 1607 cm^-1 was significantly increased in cancer cells compared with normal cells. 3) Compared with normal tissues, the peak representing PO^2- symmetric stretching vibration shifted from 1088 cm^-1 to 1083 cm^-1 in cancer tissue, and the characteristic peak for collagen at 938 cm^-1 shifted to 944 cm^-1. In addition, an extra characteristic peak indicating C = C stretching vibration appeared at 1379 cm^-1 in the lipid spectrum in cancer tissue.

Conclusions

The position, intensity, and shape of peaks in the Raman spectra of DNA, nuclei, and tissue from gastric cancer were significantly different compared with those of normal cells. These results indicate that the DNA phosphate backbone becomes unstable in cancer cells and might be broken; the relative content of histones is increased and stable; the relative collagen content is reduced, facilitating cancer cell metastasis; and the relative content of unsaturated fatty acids is increased, increasing the mobility of the plasma membrane of cancer cells.     

Raman spectroscopy of DNA packaging in individual human sperm cells distinguishes normal from abnormal cells

Healthy human males produce sperm cells of which about 25–40% have abnormal head shapes. Increases in the percentage of sperm exhibiting aberrant sperm head morphologies have been correlated with male infertility, and biochemical studies of pooled sperm have suggested that sperm with abnormal shape may contain DNA that has not been properly repackaged by protamine during spermatid development. We have used micro‐Raman spectroscopy to obtain Raman spectra from individual human sperm cells and examined how differences in the Raman spectra of sperm chromatin correlate with cell shape. We show that Raman spectra of individual sperm cells contain vibrational marker modes that can be used to assess the efficiency of DNA‐packaging for each cell. Raman spectra obtained from sperm cells with normal shape provide evidence that DNA in these sperm is very efficiently packaged. We find, however, that the relative protein content per cell and DNA packaging efficiencies are distributed over a relatively wide range for sperm cells with both normal and abnormal shape. These findings indicate that single cell Raman spectroscopy should be a valuable tool in assessing the quality of sperm cells for in‐vitro fertilization. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Effects of reduced calcium on proliferation and cell viability in tumorigenic and nontumorigenic rat tracheal epithelial cell lines

We have compared the in vitro growth and viability of tumorigenic and nontumorigenic rat tracheal epithelial cell lines over a range of calcium concentrations from 0.003 to 0.85 mM. A greater dependence on calcium for proliferation was seen in the nontumorigenic line as compared to the tumorigenic line at both the colony formation level and in mass cultures. In the latter culture condition, a marked differential effect on cell survival was also demonstrated. These differences in calcium dependence were seen in media containing fetal bovine serum or low concentrations of newborn calf serum and in a serum-free medium developed for these cells. The effect was also independent of the method used for calcium removal i.e., either by chelex treatment or the inclusion of EGTA. Therefore, loss of calcium dependence may be associated with tumorigenicity in rat tracheal epithelial cells offering a selectable marker for neoplastic cells in carcinogen-exposed preneoplastic cell populations.     

Computer-assisted imaging cytometry of nuclear chromatin reveals bone tumor virus infection and neoplastic transformation of adherent osteoblast-like cells

Established osteoblast-like (OB) cells infected with the bone tumor-inducing C-type retrovirus OA MuLV remained nontumorigenic over 104 cell culture passages. DNA histograms revealed a new cell population with a stem line peak at 5c. A second OA MuLV-infected OB cell line underwent neoplastic transformation with increasing passage level. These cells showed diffuse aneuploidy. Stepwise linear discriminant analysis of the chromatin structure of control, OA MuLV-infected, and FBR osteosarcoma virus-transformed cell lines resulted in various levels of discrimination ranging between 79.6% for control cells versus nontumorigenic OA MuLV-infected cells, and 96.6% for nontumorigenic OA MuLV-infected cells versus FBR osteosarcoma virus-transformed cells. OA MuLV-infected tumorigenic cells and FBR osteosarcoma virus-transformed cells were discriminated at a 93.6% level.     

DNA repair in mouse embryo fibroblasts. II. Responses of nontransformed, preneoplastic and tumorigenic cells to ultraviolet irradiation

Ultraviolet light-induced excision repair, as measured by single-strand DNA-break accumulation in the presence of hydroxyurea and 1-beta-D-arabinofuranosylcytosine, undergoes an apparent decline concomitant with spontaneous transformation of mouse cells in vitro. This decline is seen in preneoplastic transformed cells as well as tumorigenic cells, suggesting that it is an early event in transformation. The difference between nontransformed and transformed mouse cells in apparent incision rates is greatest at short times after irradiation when nontransformed cells show a transient phase of rapid incision. No gross differences in the effects of UV on replicative DNA synthesis, bulk RNA synthesis, cell proliferation or clonal survival in nontransformed and transformed cells were seen, in spite of the reduced incision capacity of the latter. Taken together the results suggest that transformed cells are capable of growth in the presence of significantly increased amounts of DNA damage. A decreased ability of nontumorigenic cells to remove DNA lesions, coupled with unrestricted growth, may be responsible for genetic alterations which increase the probability of a cell becoming tumorigenic.     

An improved DAPI-DNA cytofluorometry by hypotonic cytolysis. Its application to tumor cell heterogeneity

The effect of cell cytolysis on DAPI-DNA cytofluorometry was studied in order to eliminate nonspecific cytoplasmic hindrances in DAPI staining. A great improvement in the reproducibility of DNA determination was attained when cells were treated with hypotonic KCl (0.075 M) solution. The DNA content per cell of the mgC5 clonal line (normal diploid cell containing 2C DNA units) after hypotonic treatment was 3.07 +/- 0.06, while that of mgC5 cells without hypotonic treatment was 3.50 +/- 0.26, indicating a remarkable decrease from 7.4% to 2.0% in the coefficient of variation. In detecting tumor cell heterogeneity, our cytolytic method was superior to the method of counting the mode of chromosome number. The modal chromosome numbers of nontumorigenic m and its derivatives, mgC4 and mgC5, were 69, 68 and 67 respectively, while those of their tumorigenic counterparts, magc, mgC4V1 and mgC5V1 were 68, 66 and 67. The DNA contents of nontumorigenic m, mgC4 and mgC5 were 3.00 +/- 0.10 (mean +/- SD), 3.07 +/- 0.06 and 3.07 +/- 0.12, while those of their tumorigenic counterparts, magc, mgC4V1 and mgC5V1 were 3.67 +/- 0.15, 3.23 +/- 0.06, 3.27 +/- 0.06 respectively. A statistically significant difference in DNA content between the tumorigenic and nontumorigenic cells was observed at least at the p less than 0.05 level (t-test) when DNA content was employed for comparison, but not when the modal chromosome number was employed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell-cycle-dependent variations in FTIR micro-spectra of single proliferating HeLa cells: principal component and artificial neural network analysis

We have previously reported spectral differences for cells at different stages of the eukaryotic cell division cycle. These differences are due to the drastic biochemical and morphological changes that occur as a consequence of cell proliferation. We correlate these changes in FTIR absorption and Raman spectra of individual cells with their biochemical age (or phase in the cell cycle), determined by immunohistochemical staining to detect the appearance (and subsequent disappearance) of cell-cycle-specific cyclins, and/or the occurrence of DNA synthesis. Once spectra were correlated with their cells' staining patterns, we used methods of multivariate statistics to analyze the changes in cellular spectra as a function of cell cycle phase.