Flow cytometric immunofluorescence assay for quantification of cyclobutyldithymine dimers in separate phases of the cell cycle

Quantitative immunofluorescence assays for the measurement ofcyclobutyldithymine dimers (T < > T) based on computer-assistedimmunofluorescence microscopy have recently been described.Here we present a modified assay for T < > T based onflow cytometry. This method has the advantage that T < >T can be quantified in separate phases of the cell cycle bythe fluorescent counterstaining of nuclear DNA and subsequentselection on DNA content. The H3 monoclonal antibody directedat T < > T binds to partially denatured DNA in situ. Theantibody is labeled with fluorescein isothiocyanate (FITC) andDNA is stained with the intercalating dye 7-amino-actinomycinD. FITC fluorescence increases linearly with dose of UV-C radiation(up to 45 J/m²) of cultured human fibroblasts. A linear fluorescence—doserelationship was also found for epidermal cells of SKH:HR1 hairlessmice after in vivo irradiation with UV-B (FS40 sunlamp, up to3750 J/m²). This technique allows a quick assessment of UV damagelevels in 10 000s of cells and makes immunofluorescence of DNAdamage more accessible to other research groups.     

Single-cell immunofluorescence assay for terminal transferase: Human leukaemic and non-leukaemic cells

The characteristics of a single-cell immunofluorescence assay for terminal deoxynucleotidyl transferase (terminal transferase, TdT) is described. The data indicate that the single-cell immunofluorescence assay is highly efficient and specific for the detection of cells containing TdT. Using this assay, we have examined 124 marrow or peripheral-blood samples from 104 patients with or without haematological malignancies. Results indicate that TdT⁺ cells from 6% to 100% were found in the following patients: 34/40 samples from patients with ALL at the time of diagnosis or during relapse; 2/3 patients with acute undifferentiated leukaemia; 2/3 patients with acute myelomonocytic leukaemia; 1/24 patients with acute myeloblastic leukaemia; 1/5 patients with chronic myelocytic leukaemia (CML) in blastic crisis; and 2/2 patients with diffuse lymphoblastic lymphoma. In contrast less than 1% of TdT⁺ cells were found in 20 marrow or peripheral-blood samples from ALL patients in complete remission; 8 patients with CML in chronic phase; 2 patients with myeloma; 1 sample from a patient with Hodgkin''s disease, peripheral-blood samples from 7 normal donors and marrow samples from 6 patients without haematological malignancies. TdT⁺ cells were also found in association with cells with lymphoblast morphology. The TdT⁺ cells in marrow were shown to be directly correlated with the percentage of morphological lymphoblasts, with a Spearman rank coefficient of 0·81, significant at a 0·001 level. In 2 longitudinal studies of 2 ALL patients with TdT⁺ cells at diagnosis, the percentage TdT⁺ cells also changed in parallel with the proportion of lymphoblasts. However, studies of 2 other patients with morphologically diagnosed ALL with < 1% TdT⁺ cells at diagnosis also showed < 1% TdT⁺ cells throughout the period studied, indicating a stable phenotype of blast cells in these patients. The single-cell immunofluorescence assay for TdT, which requires < 0·1% of the cells used in a conventional biochemical assay, is highly specific, and could provide a technically more efficient alternative for use in clinics as well as in experimental investigations of subpopulations of leukaemic and normal marrow cells.     

Quantitative assay for carcinogen altered differentiation in mouse epidermal cells

Basal epidermal cells can be selectively maintained as a monolayer in culture medium containing a low ionic calcium concentration of 0.01-0.10 mM. Cessation of proliferation, maturation and shedding of squamous sheets can be induced in this population by increasing the calcium concentration above 0.1 mM. Since alterations in the regulation of proliferation and differentiation are associated with epidermal carcinogenesis in vivo, it appeared reasonable that changes in the phenotypic response to calcium might follow exposure to carcinogens in vitro. Support for this hypothesis was provided by the observation that malignant epidermal cells continued to proliferate when switched from low to high calcium medium, and could thus be selected from a mixture of such cells and a large excess of normal cells which did not survive after induced differentiation. Normal primary epidermal cells were plated in low calcium medium, treated on day 3 with a chemical carcinogen, maintained for 3-9 weeks in low calcium (0.02 mM) and then switched to high calcium medium (1.4 mM). After an additional 4 weeks, surviving epithelial colonies were fixed, stained with rhodamine and counted. Treatment of cultures with 7,12-dimethylbenz[a]anthracene or N-methyl-N'-nitro-N-nitrosoguanidine yielded 4-10 fold more colonies than solvent controls. Colony number was proportional to carcinogen dose for both agents, and increased with time in low calcium prior to selection by calcium increase. Cells obtained from colonies in treated cultures demonstrated characteristic epidermal morphology and keratinization, and could be subcultured, but did not grow in agar or produce tumors in syngeneic hosts. This model system represents a quantitative assay for carcinogen altered epithelial cell differentiation and may select for an early property of preneoplastic epidermal cells.     

Rapid fluorescence-based assay for radiosensitivity and chemosensitivity testing in mammalian cells in vitro

An efficient and rapid cytotoxicity assay has been developed, particularly for radiobiological studies, utilizing 96-well microtiter plates. Several days after treatment, cell numbers per well were measured by fluorescent intensity using an automatic reader after staining with the DNA specific dye Hoechst 33258. For radiobiological applications, a microtiter plate irradiation box was designed and built which allowed a variable number of wells (minimum 4, maximum 16) to be irradiated at one time. In this manner, complete dose-response curves could be obtained from one plate. The assay depends on the growth of surviving and untreated cells, and by appropriate choice of conditions (cell numbers plated, time of assay), cell survival curves for this quick fluorescence assay were in reasonable agreement with those from a clonogenic assay for cisplatin and X-ray-induced cell killing. The assay can span 1.5-2 decades of cell survival and is suitable for any cell line which grows as a monolayer. Radiobiological applications were tested using agents or conditions which modified radiation damage. Firstly, sublethal damage repair could be demonstrated in RIF1 mouse tumor cells by comparing the survival curve for a single X-ray dose with that for two fractions separated by 4 h. Secondly, incorporation of 5-iodo-2'-deoxyuridine into cellular DNA was shown to radiosensitize Chinese Hamster cells, with similar enhancement ratios obtained from the fluorescence and clonogenic assays. Thirdly, radiosensitization by cisplatin and radioprotection by cysteamine could be readily measured using the quick fluorescence assay. The ability to have multiple dose groups per plate makes it an efficient assay for both radiosensitivity and chemosensitivity testing.     

Increase in viral antigen in individual polyoma-virus-infected mouse kidney cells as studied by quantiative immunofluorescence

Polyoma-virus infection of mouse kidney cells in vitro was studied on the single cell level by quantitative cytochemical techniques. Cells were fixed in acetone at different times after infection and reacted with fluorescein-conjugated mouse anti-polyoma-virus immune serum. Visual determinations were made as to whether the cells were fluorescent (“positive”) or not (“negative”). Visually the fluorescence was located in the nuclei in the “positive” cells. The fluorescence intensities of individual cell nuclei were then measured in a microspectrofluorimeter. Nuclei with considerable fluorescence intensity were noted to be visually “positive”. However, nuclei of cells which were usually judged to be “doubtful” could also be shown to have measurably greater fluorescence intensity than control non-infected cells. Increase in fluorescence values were noted 6-11 h after infection whereas hemagglutination tests did not become positive until 10-20 h later. With increasing time post infection, the fraction of fluorescence positive cells increased both as judged visually and as determined by the microfluorimeter. The variation in fluorescence intensities among different nuclei also increased and, 48-50 h after infection, the ratio between the highest and the lowest fluorescence values of positive cells was about 10:1. Higher fluorescence intensities were found in cytoplasms of cells with “positive” nuclei than in cells with “negative” nuclei. The cytoplasmic and nuclear fluorescence intensities correlated well in cells with “positive nuclei”. The polyoma-virus infection-induced stimulation of DNA synthesis was demonstrated by increased incorporation of ³H-thymidine and by increased amounts of DNA in the cells as measured by Feulgen microspectrophotometry. The amount of DNase-resistant DNA (i.e. viral DNA) correlated well with the intensity of immunofluorescence measured on the same individual nuclei of infected cells. This was taken as one indication that viral antigens can be measured on the single cell level by quantitative immunofluorimetry.     

Analysis of anti-HTLV-I antibody by strip radioimmunoassay--comparison with indirect immunofluorescence assay, enzyme-linked immunosorbent assay and membrane immunofluorescence assay

Antibodies against human T-cell lymphotropic virus type I (HTLV-I) in the sera from 60 patients with adult T-cell leukemia and 21 carriers who were suspected of having HTLV-I infection were investigated by indirect immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA), membrane immunofluorescence assay (MIA) and strip radio immunoassay based on the western blotting technique (SRIA). The sera of 2 of the carriers who were seropositive in IFA and ELISA were negative in MIA and did not react with virus-specific proteins by SRIA. Two sera were negative in IFA and ELISA. These sera were positive in MIA and reacted with only the envelope-related glycoprotein (gp46) and not with gag-related proteins (p28, p24, p19) by SRIA. These findings suggest that the main antigens defined by IFA and ELISA are gag-related proteins and some sera which do not contain anti-HTLV-I antibodies give false-positive results because of the reaction to unknown cellular components. Also some sera may have antibodies against only envelope glycoproteins, and these sera may give false-negative results in IFA and ELISA.     

DNA microfiltration assay: a simple technique for detecting DNA damage in mammalian cells

A simple method for detection of DNA single-strand breaks (DNA-SSB)in cultured cells is described, based on filtration of alkaline-lysedcells through microfilters. After exposure to potentially DNAdamaging agents, the cells are transferred to 0.8 µm celluloseacetate filters mounted in microfihter devices where they arewashed, lysed and centrifuged to separate undamaged DNA fromdamaged DNA. When human bronchiolar cells (l4Br) were exposedto different DNA damaging agents, hydrogen peroxide, N-methyl-N-nitroN-nitrosoguanidine or 4-nitroquinoline-1-oxide, there was goodcorrelation between the extent of DNA damage assessed by thisfiltration techniqe and by DNA precipitation assay. DNA-SSBwere also detected by the filtration technique after exposureof bronchiolar cells to phorbol ester-stimulated human neutrophils.The filtration assay is easy to perform, the sample handlingcapacity is very high, and no expensive or complicated laboratoryequipment is required. It may therefore be an alternative, ora complement, to other methods for detection of DNA-SSB.     

The bromodeoxyuridine comet assay: detection of maturation of recently replicated DNA in individual cells

The single-cell gel electrophoresis (Comet) assay is a relatively simple method of measuring DNA single strand breaks and alkali-labile sites in individual cells. We have combined this with bromodeoxyuridine (BrdUrd) labeling of DNA and immunolocalization of the BrdUrd to assess DNA replicative integrity on a single-cell basis. We show that the existence of strand discontinuities in recently replicated domains of DNA, caused during semiconservative replication or exacerbated by the arrest of replicative polymerases at UV irradiation- or chemical-induced lesions, can be detected in individual cells. Data obtained from BrdUrd-Comets are consistent with biochemical data derived with a range of techniques showing that DNA replication involves the creation of strand breaks or gaps adjacent to recently replicated material, and that DNA damage prolongs the duration of such discontinuities where DNA polymerases are stalled opposite lesions (R. T. Johnson et al, The Legacy of Cell Fusion, pp. 50-67, Oxford: Science Publications, 1994; R. B. Painter, J. Mol. Biol., 143: 289-301, 1980.). Compared with standard biochemical techniques, the BrdUrd-Comet assay is simple and suitable for the accurate and automatable assessment of replicative integrity in very small numbers of mammalian cells, such as may be obtained by biopsy.     

Repair of sulfur mustard-induced DNA damage in mammalian cells measured by a host cell reactivation assay

DNA damage is thought to be the initial event that causes sulfur mustard (SM) toxicity, while the ability of cells to repair this damage is thought to provide a degree of natural protection. To investigate the repair process, we have damaged plasmids containing the firefly luciferase gene with either SM or its monofunctional analog, 2-chloroethyl ethyl sulfide (CEES). Damaged plasmids were transfected into wild-type and nucleotide excision repair (NER) deficient Chinese hamster ovary cells; these cells were also transfected with a second reporter plasmid containing RENILLA: luciferase as an internal control on the efficiency of transfection. Transfected cells were incubated at 37 degrees C for 27 h and then both firefly and RENILLA: luciferase intensities were measured on the same samples with the dual luciferase reporter assay. Bioluminescence in lysates from cells transfected with damaged plasmid, expressed as a percentage of the bioluminescence from cells transfected with undamaged plasmid, is increased by host cell repair activity. The results show that NER-competent cells have a higher reactivation capacity than NER-deficient cells for plasmids damaged by either SM or CEES. Significantly, NER-competent cells are also more resistant to the toxic effects of SM and CEES, indicating that NER is not only proficient in repairing DNA damage caused by either agent but also in decreasing their toxicity. This host cell repair assay can now be used to determine what other cellular mechanisms protect cells from mustard toxicity and under what conditions these mechanisms are most effective.     

Quantitative and qualitative in vivo angiogenesis assay

We describe the development and optimization of an in vivo angiogenesis assay utilizing gelfoam sponges impregnated with 0.4% agarose and different proangiogenic factors, such as basic fibroblast growth factor (bFGF), vascular epidermal growth factor (VEGF), tumor growth factor-alpha (TGF-alpha), and endothelial growth factor (EGF). The sponges are implanted into the subcutis of mice and harvested after different times. The gelfoam sponges are fixed, sectioned, and stained with fluorescent antibodies against CD31. The median number of CD31+ cells is determined in 10 different 0.159-mm2 fields. Proangiogenic molecules induced significant migration and proliferation of endothelial cells. To demonstrate the utility of this assay for evaluation of an antiangiogenic agent, mice were implanted with gelfoam sponges containing different proangiogenic factors and treated orally with water or PTK 787, a novel tyrosine kinase inhibitor with specific activity against the VEGF-R. PTK 787 significantly inhibited angiogenesis in sponges containing agarose + VEGF but not other proangiogenic molecules. The data show that the implanted gelfoam sponges provide a reliable quantitative assay to study in vivo angiogenesis.     

Quantitative assay for evaluating immunocompetence and DNA repair capacity

Incorporation of radioactive thymidine into newly synthesized DNA is the basis of an assay frequently used to study immunosuppression in cancer patients. It has also been used to measure the amount of excision repair performed by non-replicating cells damaged by carcinogens. For human lymphocytes (and probably other cell types), these assays are unreliable as they are currently being performed. We report a modified assay that allows accurate comparisons of the immunocompetence and DNA repair capacity of different individuals. With this assay, cells can be studied in their autologous plasma and the role of biological response modifiers can be assessed.     

Evidence for two states of thermotolerance in mammalian cells

The effect of the inhibition of protein synthesis on the development of thermotolerance in Chinese hamster fibroblasts following a brief heat shock or exposure to sodium arsenite has been examined. Under conditions that inhibit protein synthesis by 95 per cent, significant amounts of thermotolerance develop after a brief exposure to 45°C or continuous exposure to 41°C, without the significant accumulation of heat shock proteins. However, no thermotolerance development in cells treated with sodium arsenite was observed if protein synthesis was inhibited. Heated cells which developed thermotolerance in the absence of protein synthesis are subject to the thermal sensitizing action of a subsequent exposure to amino acid analogues, while cells which developed thermotolerance with unimpeded protein synthesis are refractory. These results suggest that heat can simultaneously induce two states of thermotolerance, only one of which is dependent on protein synthesis. These two states can be distinguished operationally with respect to their response to amino acid analogue exposure.     

Evidence for two states of thermotolerance in mammalian cells

The effect of the inhibition of protein synthesis on the development of thermotolerance in Chinese hamster fibroblasts following a brief heat shock or exposure to sodium arsenite has been examined. Under conditions that inhibit protein synthesis by 95 per cent, significant amounts of thermotolerance develop after a brief exposure to 45 degrees C or continuous exposure to 41 degrees C, without the significant accumulation of heat shock proteins. However, no thermotolerance development in cells treated with sodium arsenite was observed if protein synthesis was inhibited. Heated cells which developed thermotolerance in the absence of protein synthesis are subject to the thermal sensitizing action of subsequent exposure to amino acid analogues, while cells which developed thermotolerance with unimpeded protein synthesis are refractory. These results suggest that heat can simultaneously induce two states of thermotolerance, only one of which is dependent on protein synthesis. These two states can be distinguished operationally with respect to their response to amino acid analogue exposure.     

Guanine ribonucleotide depletion in mammalian cells

Summary In a previous report we demonstrated in mouse lymphoma (S-49) cells that DNA synthesis inhibition resulting from guanine starvation is associated with GTP rather than dGTP depletion. Since several effective anticancer drugs act via guanine depletion, it is imporatant to test whether critical GTP depletion is unique to S-49 cells or also occurs in other cell lines. Mycophenolic acid-induced guanine starvation caused a drastic DNA synthesis inhibition in the human lymphoblastic T leukemia (CEM) and the mouse B leukemia (L1210) cell lines, which was again associated with GTP depletion rather than dGTP depletion. These results suggest that GTP depletion represents a common target of purine antimetabolites in mammalian cells.     

Excision repair is required for genotoxin-induced mutagenesis in mammalian cells

Certain hexavalent chromium [Cr(VI)] compounds are human lung carcinogens. Although much is known about Cr-induced DNA damage, very little is known about mechanisms of Cr(VI) mutagenesis and the role that DNA repair plays in this process. Our goal was to investigate the role of excision repair (ER) pathways in Cr(VI)-mediated mutagenesis in mammalian cells. Repair-proficient Chinese hamster ovary cells (AA8), nucleotide excision repair (NER)-deficient (UV-5) and base excision repair (BER)-inhibited cells were treated with Cr(VI) and monitored for forward mutation frequency at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus. BER was inhibited using methoxyamine hydrochloride (Mx), which binds to apurinic/apyrimidinic sites generated during BER. Notably, we found that both NER-deficient (UV-5 and UV-41) and BER-inhibited (AA8 + Mx) cells displayed attenuated Cr(VI) mutagenesis. To determine whether this was unique to Cr(VI), we included the alkylating agent, methylmethane sulfonate (MMS) and ultraviolet (UV) radiation (260 nm) in our studies. Similar to Cr(VI), UV-5 cells exhibited a marked attenuation of MMS mutagenesis, but were hypermutagenic following UV exposure. Moreover, UV-5 cells expressing human xeroderma pigmentosum complementation group D displayed similar sensitivity to Cr(VI) and MMS-induced mutagenesis as AA8 controls, indicating that the genetic loss of NER was responsible for attenuated mutagenesis. Interestingly, Cr(VI)-induced clastogenesis was also attenuated in NER-deficient and BER-inhibited cells. Taken together, our results suggest that NER and BER are required for Cr(VI) and MMS-induced genomic instability. We postulate that, in the absence of ER, DNA damage is channeled into an error-free system of DNA repair or damage tolerance.