Chromosomal damage in two X-ray irradiated cell lines: influence of cell cycle stage and irradiation temperature.

UNLABELLED: The aim of this study was to investigate if irradiation with X-rays in different cell cycle phases resulted in a different response as measured with the micronucleus technique. In addition, the influence of irradiation temperature was investigated. MATERIALS AND METHODS: Cells from a non-transformed human fibroblast cell line, HS2429, and a human breast cancer cell line, MCF-7, were synchronized by thymidine block and irradiated at either 2 degrees C or 37 degrees C in the G1-, S- and G2/M-phases. After cytokinesis-block by cytochalasin B, the frequency of micronuclei was determined. RESULTS: Clear dose-response relationships were found. More micronuclei were detected in fibroblast cells irradiated in G1 and S than in G2/M, while the differences were not as prominent in MCF-7 cells. The irradiation temperature had no significant influence on the formation of micronuclei in either of the cell lines. CONCLUSION: The formation of micronuclei varies with the cell cycle stage at the time of irradiation.

The biological effect of pentoxifylline on the survival of human head and neck cancer cells treated with continuous low and high dose-rate irradiation.

AIM: The aim of this study was to compare the radiosensitivity effect of the G2/M arrest-abrogating substance, pentoxifylline (PTX), with high dose-rate irradiation (HDRI) and low dose-rate irradiation (LDRI), during which DNA repair and cell proliferation occur. METHODS: Three squamous cell carcinoma cell lines, FaDu, RPMI 2650 and SCC-61, with differences in genomic imbalance and intrinsic radiosensitivity, were irradiated with 140 cGy/min (HDRI) and 0.7 cGy/min (LDRI) in the presence and absence of 2.0 mM PTX. The surviving fraction at 2.0 Gy (SF2) and cell-cycle phase distribution were assessed by DNA flow cytometry analysis and bromodeoxyuridine incorporation. RESULTS: With HDRI and LDRI the SF2 of FaDu cells decreased by 38.5% and 27.6%, respectively, while the corresponding figures for RPMI 2650 were 28.5% and 48.5%, and for SCC-61 were 44.2% and 28.6%. Increases in G2 populations were evident after both HDRI and LDRI of all cell lines. CONCLUSIONS: The enhancement in the cytotoxic effect of PTX was statistically significant after HDRI as well as after LDRI in all three cell lines. We therefore conclude that PTX in combination with LDRI is worth further study, both in vitro, for disclosing underlying mechanisms, and in vivo, to confirm the findings.

Cell growth kinetics of the human cell line Colo-205 irradiated with photons and astatine-211 alpha-particles.

Cell growth kinetics following Astatine-211 (211At, alpha-particle emitter) and photon irradiation were studied for the human colorectal cell line Colo-205. A growth assay using 96-well plates was chosen. The growth kinetics could be simulated by assuming certain fractions of cells with various proliferative capacities, i.e. from none up to 5 cell doublings, in addition to the defined survivors with remaining unlimited clonogenic capacity. No significant difference in cell growth characteristics was seen between 211At and photon irradiation. The cell doubling time, as calculated from the increment in optical density, was compared with the results from BrdU experiments in the early phases of growth (Tpot = 18.5 +/- 0.6 h for LDR (low dose rate) photon irradiated and 20.3 +/- 0.8 hours for sham-irradiated cells 40-45 hours post-irradiation) confirming the transient accelerated growth of irradiated cells. No statistically significant difference in growth was found between LDR, MDR (medium dose rate) and HDR (high dose rate) photon irradiation.

In vitro effects of free 211At,211At-albumin and 211At-monoclonal antibody compared to external photon irradiation on two human cancer cell lines.

BACKGROUND: The aim of this study was to perform various 211At irradiations of importance for the evaluation of 211At-radioimmunotherapy, and compare the effect with that of low linear energy transfer (LET) radiation. MATERIALS AND METHODS: All irradiations were performed on low-concentration single-cell suspensions. Growth assays using 96-well plates were used to estimate apparent cell survival. Centrifuge tube filters were used to estimate the cell uptake and binding of 211At. RESULTS: A relative biological effect (RBE) of 12 +/- 2 (Colo-205) and 5.3 +/- 0.7 (OVCAR-3) was found from 211At-albumin irradiations. There was a 174 +/- 28 times higher free 211At concentration in the cell fraction than in the surrounding medium. For 211At-MAb, an 8,000-30,000 times higher concentration in the cell fraction was achieved, compared to the medium. Corrected for the uptake, an average of 31 +/- 2 ([211At]-astatine) or 26 +/- 5 ([211At]-MAb) decays per cell were required for 37% survival of Colo-205 cells. An average of 19 +/- 3 decays ([211At]-astatine) were required per OVCAR-3 cell. CONCLUSIONS: Cell uptake and binding of 211At was unexpectedly high, possibly favouring its therapeutic use. The binding is probably to the cell surface. The RBE is 5.3 +/- 0.7 for OVCAR-3 and 12 +/- 2 for Colo-205 cells.

S-phase fraction related to prognosis in localised prostate cancer. No specific significance of chromosome 7 gain or deletion of 7q31.1.

A flow-cytometric (FCM) and fluorescence in situ hybridization (FISH) study was performed in 153 patients with clinically localised prostate cancer (PC) to evaluate retrospectively the prognostic significance of DNA ploidy, S-phase fraction (SPF) and chromosome 7 copy number. Deletions in 7q31.1 were analysed in a subset of 26 tumours. The mean follow-up time was 6 years (range 4-16 years). Twelve cases of benign prostatic hyperplasia (BPH) were studied as a control. Chromosome 7 enumeration and deletion studies were conducted using the alpha-satellite D7Z1 probe and a cosmid probe specific for the marker D7S522 on 7q31.1. Higher SPF was associated with shorter overall survival and shorter time to local progression and metastasis. Near diploid (DNA index 1.05-1.20) cases had a lower frequency of metastases and lower Gleason scores than aneuploid cases. Increased absolute chromosome 7 copy number (centromere count) was associated with higher Gleason score, higher SPF and shorter local progression-free and prostate cancer survival. Absolute chromosome 7 copy number was concordant with FCM DNA ploidy in the majority (75%) of cases. Relative gain or loss of chromosome 7 (centromere counts compared to ploidy) was infrequent, and no correlation was found with clinical parameters. Deletions in 7q31.1 were infrequent. Our results indicate that in localised PC (i) SPF is a prognostic factor, (ii) absolute chromosome 7 copy number is concordant with the ploidy status of the tumour (relative gain or loss of chromosome 7 is infrequent and has no independent prognostic value) and (iii) the frequency of deletions in 7q31.1 is low and not correlated with clinical outcome.

Effects of the alpha-particle emitter At-211 and low-dose-rate gamma-radiation on the human cell line Colo-205 as studied with a growth assay.

BACKGROUND: The aim of this study was to investigate the biological effect of the alpha-particle-emitting isotope astatine-211 on the human cell line Colo-205 and to compare it with that of low-dose-rate gamma-radiation. MATERIALS AND METHODS: Plastic (PMMA) rotating phantoms were constructed, allowing precise dosimetry on a cellular level for both types of radiation. Growth assays using 96-well plates were used to estimate apparent cell survival for the two types of radiation. From this, the relative biological effect (RBE) could be estimated. RESULTS: Irradiation of the cells with 211At resulted in an RBE of 25.1 +/- 6.7 at 37% survival, and 17.3 +/- 2.5 at 10% survival, when compared with low-dose-rate gamma-irradiation. The absorbed dose at 37% survival, 0.12 Gy, corresponds to 2.2 traversals of alpha-particles through the cell nuclei. For cells irradiated with gamma-radiation (1 and 2 Gy), an apparent cell survival above unity was observed up to 50 hours post-irradiation, indicating a possible radiation hormesis effect. CONCLUSIONS: The RBE of 211At found in this growth-assay study was significantly higher than previously presented values. The difference might be due to the use of low-dose-rate gamma-radiation as reference. The RBE presented here could prove valuable when evaluating 211At-labelled compounds for radiotherapy.

Opposing effects of suramin and DL-alpha-difluoromethylornithine on polyamine metabolism contribute to a synergistic action on B16 melanoma cell growth in vitro.

Polyamines are crucial for normal and neoplastic cell growth. Treatment with the polyanionic drug suramin has pronounced antigrowth activity in several tumor cell lines, but its clinical use has been hampered by its toxicity. We have earlier shown that suramin affects cellular polyamine metabolism and transport, and that these effects were, in some respects, opposite to those of alpha-difluoromethylomithine (DFMO), a specific inhibitor to ornithine decarboxylase, a key metabolic enzyme for polyamines. DFMO has been used in anticancer trials, although with limited success. Combinations of suramin and DFMO were, hence, evaluated in vitro and were found to strongly inhibit B16 melanoma cell proliferation. DFMO alone induced melanoma cell differentiation, and suramin used in combination with DFMO did not abrogate this DFMO-induced differentiation. Synergy analysis demonstrated a pronounced growth-inhibitory synergism between suramin and DFMO. The results suggest that the efficacy of combinations of DFMO with suramin or its analogues should be further explored, especially in cells requiring high levels of polyamines for their growth.

Effects of suramin on polyamine metabolism in B16 murine melanoma cells.

Polyamines and their biosynthetic enzymes, such as ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), are crucial for normal and neoplastic cell growth and differentiation. Suramin inhibits the growth of several tumor cells by affecting various intracellular targets, but its effects on polyamines are not known. In this study, the effects of suramin on some parameters of polyamine metabolism in B16 melanoma cells were investigated in vitro. Suramin increased cellular ODC activity and ODC mRNA levels, whereas the drug was directly inhibitory to the enzyme. AdoMetDC was not affected. Cellular putrescine levels were enhanced by suramin, whereas spermidine and spermine pools were unaltered. Cells cultured in the presence of suramin showed decreased cellular polyamine transport, but no direct inhibitory effect on the polyamine transporter could be found. Fluorescence spectroscopy demonstrated a direct interaction between suramin and spermine. It may be concluded that suramin affects polyamine metabolism, and that its effects in some respects are opposite to those of alpha-difluoromethylomithine (DFMO), a specific inhibitor of ODC.

Tumour growth and cell kinetics in variants of a human endometrial adenocarcinoma expressing either wild-type or mutant p53.

We have compared the baseline cell proliferation and tumour growth in two variants of a human endometrial adenocarcinoma grown in nude mice. One of these tumour variants expressed wild-type p53 whereas the other had mutations of the p53 gene at codon 175 in both alleles and at codon 248 in one allele. There was no difference in growth rate between the tumour variants. Cell proliferation parameters, such as labelling index and S-phase fraction, were significantly increased in the tumour with mutated p53 and consequently there was a significantly lower proportion of cells in the G1-phase, proposing an at least partial loss of suppressor function in this tumour. Semi-quantitative analysis of the p53 and bcl-2 proteins showed a significant overexpression of p53 and a decreased expression of the bcl-2 protein in the p53 mutated tumour variant compared with the variant with wild-type p53. We conclude that wild-type p53 protein acts as an active suppressor in the regulation of the baseline growth and cell kinetics of this tumour and could be linked through a p53--bcl-2 system in human endometrial adenocarcinomas.

Short- and long-term oestradiol treatment inhibits growth and alters expression of p21WAF1/Cip1 in an endometrial adenocarcinoma cell line lacking functional p53.

Oestrogen is assumed to play a significant role in cell cycle regulation of cells expressing the oestrogen receptor, although its mechanism of action is not yet well defined. To examine this, a mutant p53-expressing human endometrial adenocarcinoma cell line of the oestradiol-inhibited growth phenotype was treated with oestradiol for 2 weeks (short-term) and 6 months (long-term). With short-term treatment, cells were treated with increasing doses of oestradiol. The highest dose, 1 microM, was used in the long-term interval. The influence of the hormone on growth, proliferation and expression of some cell cycle and apoptosis-related proteins was evaluated. In cells treated for 2 weeks, there was a dose-dependent inhibition of both growth and proliferation with a significant decrease in labelling index (LI) and S-phase fraction (SPF) and a simultaneous increase in the fraction of cells in G0/G1. Extending the oestradiol treatment to 6 months showed further growth retardation and decreased proliferation with cells accumulating in G0/G1. Analysis of the expression of p21WAF1/Cip1 showed a nearly 2-fold increase after 2 weeks treatment with 1 microM oestradiol, which was also observed after long-term treatment without any further increase in protein levels. Expression of the anti-apoptotic protein bcl-2 was not affected after short-term treatment but decreased significantly after 6 months treatment compared to control cells. Our results suggest the existence of a p53-independent pathway of oestradiol regulation of growth and proliferation in this human endometrial adenocarcinoma, resulting in accumulation of cells in G0/G1 through p21WAF1/Cip1 induction and, after prolonged treatment, downregulation of bcl-2 protein.

A rapid and simplified technique for analysis of archival formalin-fixed, paraffin-embedded tissue by fluorescence in situ hybridization (FISH).

We here present a simplification of the entire procedure for preparing formalin-fixed, paraffin-embedded tissue to be used for FISH-analysis. The steps for deparaffinisation and disintegration of the tissue to produce intact cell nuclei in a monodispersed suspension are detailed as well as the hybridisation steps. The procedure results in a clear cell suspension with intact cell nuclei and without clumped debris particles. The enzymatic digestion is restricted to the trypsinisation step of the deparaffinisation procedure. No further pretreatments prior to hybridization are performed. This modified technique has been successfully applied to different formalin-fixed, paraffin-embedded materials.

Estradiol regulates tumor growth by influencing p53 and bcl-2 expression in human endometrial adenocarcinomas grown in nude mice.

This study analyzes the effects of estradiol on p53 and bcl-2 expression, tumor growth and cell kinetic parameters in three human endometrial adenocarcinomas grown in nude mice. The tumors used were estradiol receptor (ER) positive but differed in receptor concentration and hormone sensitivity. All three tumors expressed wild-type p53 protein. Using a tumor with an estradiol independent but responsive (inhibited) growth phenotype, we found that an increase in the circulating estradiol concentration led to increases in p53 expression and a decrease in bcl-2 levels, resulting in increased cell loss (CL) measured as delayed tumor growth. In another tumor which demonstrated estradiol independent and resistant growth, we observed an estradiol dose-related increase in p53 expression but no changes in bcl-2 expression or cell kinetic parameters. The ER mechanism of these cells was at least partly intact, as evidenced by maintained PgR induction. The third tumor showed an estradiol independent and resistant growth phenotype and a non-functional ER mechanism, lacking PgR induction. After estradiol treatment of the tumor-bearing animals no changes were observed in p53 or bcl-2 expression or in cell kinetics. We conclude that estradiol may regulate tumor growth in some ER positive human endometrial adenocarcinomas through regulation of p53 expression, which in turn regulates the bcl-2 protein concentration. Furthermore, this regulation of p53 expression is estradiol dose dependent. These growth regulating functions appear to be strongly influenced by ER mechanisms and do not seem to operate synchronously in tumors with an estradiol resistant growth phenotype.

Characterisation of a human endometrial adenocarcinoma established in vivo and in vitro.

A moderately differentiated human endometrial adenocarcinoma was heterotransplanted into nude mice and later established as a continuous in vitro cell line. Western blot analysis showed an accumulation of p53 protein in the cell line compared to the original tumour and heterotransplants. Sequential analysis of the p53 gene revealed point mutations in codons 175 and 248 in the cell line while no mutations were found prior to in vitro establishment. Immunohistochemistry confirmed the epithelial origin of the heterotransplants and of retransplants of the cell line. The intraperitoneal retransplants remained moderately differentiated, whereas subcutaneous retransplants became less differentiated. Heterotransplants were estrogen receptor (ER) positive and progesterone receptor (PgR) negative, indicating preservation of normal steroid receptor status. The ER could not be detected in the in vitro cell line using an enzyme immunoassay, but was detected with Western blot using a polyclonal antibody toward the carboxy terminus. After estradiol treatment, the in vitro cell line became weakly positive for the PgR, suggesting the ER mechanism was at least partly intact. Tumour growth in vivo was independent of endogenous estrogen but was inhibited when the tumour-bearing animals were treated with estradiol. Analysis of cell growth kinetics by flow cytometry (FCM) after bromodeoxyuridine (BrdU)-labelling revealed no difference in S-phase fraction (SPF) or labelling index (LI) between the treated and control groups. Cell loss (CL) was significantly increased from 42% to 89%, resulting in increased tumour volume doubling time (TVDT). Under in vitro conditions estradiol treatment resulted in an increase in cell doubling time and this growth retardation was accompanied by a significant decrease in SPF and LI. The estrogen responsive (inhibited) phenotype was thus preserved in the in vitro cell line but was probably mediated through another mechanism. This cell line thus appears to represent the development of a more malignant clone with divergent receptor function and growth behaviour, and provides us with an interesting new tool for the study of tumorigenesis in the human endometrium.

Action of interferon alpha and beta on four human melanoma cell lines in vitro.

Four human melanoma cell lines with different copy numbers of chromosomes 9 and 21q, as studied by the G-band technique, fluorescent in situ hybridisation (FISH) and Polymerase chain reaction (PCR), were tested for their sensitivity to Interferon-alpha (IFN-alpha) and Interferon-beta (IFN-beta) in relation to dosage of interferon genes (#9) and interferon receptor genes (#21p). The two most sensitive cell lines were those containing the highest numbers of #9 per cell, while the number of #21q copies (receptor genes) seemed to have no influence on the interferon sensitivity.

Estradiol influences p53 expression in a human endometrial adenocarcinoma heterotransplanted into nude mice.

The influence of different estradiol concentrations on the expression of the p53 suppressor gene and on cell kinetics was examined by semiquantitative analysis of protein and bromodeoxyuridine labelling in a human endometrial adenocarcinoma grown in nude mice. We found that increasing the circulating estradiol increases (p = 0.001), and decreasing the hormone value decreases (p = 0.001) the expression of p53 in this tumor. The number of cells in the G1/G0 phase of the cell cycle was significantly higher (p = 0.03), and the number of cells in the G2/M phase was significantly lower (p = 0.01) in tumors grown in estradiol-treated mice than in tumors obtained from the nontreated group. Changes in p53 expression may possibly be explained by either altered transcription activity of the gene or increased half-life of the protein. Our results suggest an important role of estradiol in the progression of estrogen receptor (ER) positive human endometrial adenocarcinomas.

Reproducibility in DNA flow cytometric analysis of breast cancer: comparison of 12 laboratories' results for 67 sample homogenates.

Flow cytometric (FCM) DNA analysis yields information on ploidy status and the S-phase fraction (SPF), variables of prognostic importance in breast cancer. The clinical value of the SPF is currently being evaluated in prospective randomized trials. The widespread use of FCM DNA analysis emphasizes the importance of reproducibility (both intra- and interlaboratory). In this study, 67 nuclear suspensions of breast cancer samples were analyzed by 12 laboratories routinely performing FCM DNA analysis in breast cancer. No general guidelines were imposed; each laboratory used its own standard protocols. For DNA ploidy status (diploid vs. non-diploid), agreement was complete for 79% (53/67) of the samples, compared with 64% (43/67) of samples when tetraploidy was considered [i.e., euploid (diploid+tetraploid) vs. aneuploid (the remaining non-diploid)]. For the SPF, pairwise comparison of the results of all 12 laboratories yielded a mean Spearman's rank correlation of 0.78 (range: 0.54-0.93). For those 39 samples being categorized in low or high SPF by all laboratories, all agreed in 14 samples (36%). Similar patterns were obtained with kappa measures, agreement being good for ploidy status (diploid vs. non-diploid; overall kappa = 0.87 and 0.74 for euploid vs. aneuploid), but moderate for the SPF [overall kappa = 0.47 (for low SPF vs. high SPF vs. "no SPF reported")]. Discrepancies were chiefly attributable to differences in the categorization of the S-phase values, rather than in FCM procedures, other critical differences being in the detection and interpretation of near-diploid and small non-diploid cell populations, the definition of tetraploidy, and the choice and execution of the method used for S-phase estimation. Based on the observations of this study, detailed guidelines for FCM analysis and interpretation of data are proposed in the Appendix. Some issues remain, however, e.g., to standardize a method for S-phase calculation and tetraploid definition.

Protein expression of p53 and Rb suppressor genes during the first passages of human endometrial adenocarcinomas heterotransplanted into nude mice.

Five human endometrial adenocarcinomas were heterotransplanted into nude mice to study changes in p53 and Rb protein expression and loss of heterozygosity of these genes during the first nude mice passages. Three of five heterotransplanted tumours were established and measured. In one tumour over-expression of p53 protein was found in passage 1 and subsequently, whereas the original tumour did not over-express the protein. We concluded that the heterotransplantation, i.e. the new host environment, may lead to mutations in the p53 gene. Two tumours from patients and also during nude mice passages expressed p53 protein in two bands, which were probably the result of allele polymorphism. Both p53 and Rb protein expression were increased after growth in estradiol-treated animals. Our results suggest that several changes in the function of these suppressor genes, including mutations, occur during the first passages in nude mice. Consequently, the growth regulation of established tumours may differ from that in their original counterparts in the patients.

Prognostic value of DNA ploidy and S-phase fraction in endometrial cancer stage I and II: a prospective 5-year survival study.

Knowledge of reliable prognostic factors is essential in cancer treatment. Especially when intensified treatment is to be considered to improve the overall result, it is desirable to identify well-defined high-risk groups. In a prospective study DNA ploidy and S-phase fraction were measured in 88 patients with endometrial cancer stage I and II. Fresh tumor samples were analyzed using flow cytometry prior to treatment. Diploid tumors represented 84% of the cases, and aneuploid represented 16%. The mean S-phase fraction in diploid tumors was 10%, as compared with 22% in aneuploid tumors. The follow-up time was 5 years in all cases. The survival rate for patients with diploid tumors was 92% and for aneuploid tumors 36%. In the surviving patients, the mean S-phase fraction was 10.4%, a significant difference from 19.9% in the nonsurviving patients (P < 0.001). The highest mortality was found when aneuploidy was combined with an S-phase fraction over 20%, with only 11% survival for 5 years. In diploid tumors with an S-phase fraction below 20%, the survival rate was 92%. In a stepwise regression analysis, S-phase fraction was found to be of the most important prognostic value, followed by myometrial invasion and stage of the tumor and ploidy. Grade was not found to be of any important significance.

Tumor volume growth and cell proliferation in an estradiol resistant human endometrial adenocarcinoma growing in nude mice.

Ten human endometrial adenocarcinoma grafts were subsequently transplanted from an original tumour growing in nude mice for several years and nine were investigated for cell proliferation, tumour volume growth, allele imbalance and protein products of Rb and p53 suppressor genes to study a possible inherited connection between these factors. We did not find faster tumour volume growth in tumours with increased cell proliferation, in contrast to melanomas and breast cancers growing in nude mice. We concluded that a probably existing link between mechanisms regulating cell proliferation and cell loss in these two tumours does not operate in endometrial carcinoma as measured by us. The tumour progression may be one important factor which influences mechanisms regulating cell proliferation and cell loss.

Characterization of four melanoma cell lines with electron microscopy, immunocytochemistry, cytogenetics, flow cytometry, and southern analysis.

Four cell lines established from human metastatic malignant melanoma, derived from four patients, were analyzed. Ultrastructurally and immunocytochemically, the cultured tumor cells had retained characteristic features of melanocytes and of the primary malignant melanomas. The genetic stability was investigated by repeated flow-cytometric and cytogenetic analyses over 24 months of continuous cultivation. The DNA indices ranged from 1.7 to 2.1 and were stable during the entire period. The same was true for the karyotypes, which had modal numbers ranging from 50 to 84. The most common types of abnormalities were: isochromosomes i(1q), i(9q), translocations (1;17) and (3;6), and other aberrations (1p+,4p+,5p+,11p+,11q-,11q+). Abnormalities involving chromosome 1 were present in all cell lines, but loss of genetic material from chromosome 1p was demonstrated in only one of four cell lines when tested by the Southern blotting technique using a lambda MS1 probe.