Local chemical composition of nanophase-separated polymer brushes.

Using scattering scanning nearfield infrared microscopy (s-SNIM), we have imaged the nanoscale phase separation of mixed polystyrene-poly(methyl methacrylate) (PS-PMMA) brushes and investigated changes in the top layer as a function of solvent exposure. We deduce that the top-layer of the mixed brushes is composed primarily of PMMA after exposure to acetone, while after exposure to toluene this changes to PS. Access to simultaneously measured topographic and chemical information allows direct correlation of the chemical morphology of the sample with topographic information. Our results demonstrate the potential of s-SNIM for chemical mapping based on distinct infrared absorption properties of polymers with a high spatial resolution of 80 nm × 80 nm.

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.

Sensitivity to extrinsically supplied interferon and the endogenous expression of interferon in melanoma cell lines.

Interferons (IFNs) have been shown to Induce loss of growth potential in melanoma cell lines. However, human melanomas have shown limited responsiveness to clinical therapy with IFN. In a previous study on melanoma cell lines we found that greatest sensitivity to IFN was found in cell lines with the greatest number of copies of chromosome 9p, where the IFN gene family is located. In the present study the expression In melanoma cell lines of IFN genes, IFN receptor genes and standard control genes (beta-actin, glyceraldehyde-3-phosphate dehydrogenase, 18S rRNA and cyclophilin) was investigated using the reverse transcription-polymerase chain reaction, together with an exogenous standard (cyclophllin armoured RNA). We found that the sensitivity to extrinsically supplied IFN seems to correlate with the expression of endogenous IFN genes. The two melanoma cell lines producing the highest relative amount of IFN mRNA transcripts also demonstrated the most marked response to extrinsically supplied IFN. We hypothesize that tumours with enhanced endogenous IFN production may respond more positively to IFN treatment.

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.

Growth and clonogenic assays compared for irradiated MCF-7 and Colo-205 cell lines.

Clonogenic assays have been the golden standard for the assessment of cytotoxic injury from irradiation or drugs. Since such assays are time consuming, growth assays, often with automatic quantifying equipment, are frequently used. Since these procedures do not immediately reflect loss of clonogenic capacity, it was considered important to validate the two procedures using gamma-irradiation (0, 2 and 4 Gy) of two human cell lines (MCF-7 and Colo-205). The cells were growing exponentially in 96-well plates and crystal violet staining resulted in optical densities proportional to cell number. The homogeneity of optical densities within the plates was optimal if the wells to be measured were surrounded by liquid-containing ones. The slopes of the exponential growth curves were decreased upon irradiation. An "apparent cell survival", the mean of the three lowest ratios between irradiated and control cells, was defined. It was compared with the SF2 and SF4 as found in parallel Courtenay-Mills assays. In this work we found a modest underestimation of cell survival using the growth assay, ranging from 0 to 17 per cent in absolute terms.

Effects of oophorectomy on interstitial fluid pressure, blood flow and vascularity in a rat mammary tumour.

Interstitial fluid pressure (IFP), tumour size, blood flow and vascularity were measured in dimethyl-benz-alpha-anthracene-induced rat mammary tumours after oophorectomy. IFP was measured in the tumours by the wick-in-needle technique, blood flow by the labelled microsphere technique and vascularity by fluorescence vascular staining. Tumour volume was determined by measuring two diameters. Oophorectomy resulted in a decrease in tumour volume preceded by a decrease in IFP, while no significant change was observed in total tumour blood flow. The vascular cross-sectional area seemed to decrease. Cell proliferation may be of importance for the high interstitial fluid pressure in this hormone dependent tumour. A reduced metabolic requirement in the regressing tissue, down-regulating the vascular capacity, may camouflage the influence of the reduced tissue pressure on blood perfusion.

Prevalence of Klinefelter's syndrome in male breast cancer patients.

Klinefelter's syndrome (KS, XXY) as a risk factor for developing breast cancer was evaluated in a retrospective study of 93 unselected male breast cancer patients from the Healthcare region of Western Sweden. Archival normal material from lymph nodes or skin and subcutaneous tissue was examined using the FISH (fluorescence in situ hybridisation)-technique. The best yield of intact nuclei was obtained from lymph node tissue. The prevalence rate of KS in males with breast cancer was found to be 7.5 per cent, a much higher rate than previously reported (approximately 3 per cent). Methodological differences are suggested to cause the increased prevalence rate. Based on our finding and on the prevalence of KS in the normal population as well as on the incidence of MBC, a 50-fold increased risk of developing breast cancer in males with KS relative to normal males was found. The same median age at diagnosis, 72 years, was established for both groups of patients. No differences in survival were seen.

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.

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.

Vascular cross-sectional area in rat mammary tumours; influence of noradrenaline.

A double fluorescent staining technique for visualisation of blood vessels was applied to rat mammary tumours. Rats were exposed to noradrenaline or saline and the stained vascular area was recorded. In tumours exposed to noradrenaline, vascular staining was different from that in those exposed to saline, reflecting the possible closure of part of the vasculature in response to the drug.

Cytogenetic findings in 111 ovarian cancer patients: therapy-related chromosome aberrations and heterochromatic variants.

The chromosomes of 111 ovarian cancer patients were studied in G- and C-banded slides from peripheral blood lymphocyte (PBL) cultures for chromosome damage caused by chemotherapy and radiotherapy and for asymmetry of the constitutive heterochromatin of chromosomes 1, 9, and 16. We also monitored the survival of these patients to determine whether any secondary neoplasia induced by the therapy and report the findings of our investigations. Melphalan (MEL) was the only drug used in single-drug chemotherapy. The incidence of chromosome abnormalities in melphalan-treated cells (25%) was higher than in the control group (17%). The incidence of structural changes was also higher (10.5%) in the MEL-treated group than in controls (6%). After treatments with combinations of drugs, the incidence of structural changes remained at the same level (11%). In the patients receiving combined treatment with MEL and radiation, the rate of structural changes increased dramatically (24%). The overall rate of chromosome aberrations in this group was also higher (50%). Combination of two or more drugs and radiation produced only 14% structural chromosome changes. The overall rate of chromosome aberrations was also low (20%) in this group. Of 111 patients studied, only 33 were alive 6 years after initiation of the study. Of the surviving patients, eight had rearranged chromosomes in the first analysis. After 5 years, new blood samples were collected from these patients and chromosome analyses showed abnormal karyotypes in all eight patients. All chromosome abnormalities in the second analysis were completely unrelated to those in the first analysis, however. Whether the chromosome changes in the second analysis were due to therapy or to other unknown factors could not be determined. Data on C-banding and the distribution of inversions indicated that 91% of the patients had C-band heteromorphisms of chromosomes 1, 91% had heteromorphisms of chromosome 9, and 69% had heteromorphisms of chromosome 16. Furthermore, inversions were observed in chromosome 1 (41% of patients), chromosome 9 (28% of patients), and chromosome 16 (5% of patients).

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.

Heterochromatin variants in 109 ovarian cancer patients and 192 healthy subjects.

Aberrations of the C-band region of chromosome no. 1 (1qh) were studied in 109 patients with ovarian cancer and 192 healthy subjects. The groups were compared for heterochromatin size variations, intrapair size asymmetry, and inversion. No significant correlation was found between the size of 1qh and ovarian cancer. Heterochromatin size asymmetry was estimated visually and determined by objective measurement of 1qh length or area; the methods show strong correlation. The measurements were normalised by comparison with the length or area of 16p or the entire chromosome no. 1. However, since good reliability was found by simply relating the 1qh size difference to the mean 1qh size, this was considered an appropriate and simpler method of normalisation. Asymmetry indices of length and area measurements correlated well, implying that the simpler method of length measurements can be readily used. 1qh asymmetry, measured objectively or estimated visually, was significantly increased in the cancer patient group. The incidence of C-band inversion was significantly increased in the patient group. Moreover, inversion increased significantly with increasing 1qh asymmetry.

Familial occurrence of cancer and heteromorphism of the heterochromatic segment of chromosome 1.

During the last decade, evidence has been forthcoming in support of the correlation between heteromorphism of human chromosome 1qh and the incidence of various malignancies in the carriers of such heteromorphism. We present data from a family with hereditary predisposition to cancer. In this family, five members in a sibship of seven developed ovary and/or colon carcinoma at comparatively young ages. A further 4 cases of malignant disease were ascertained, when a pedigree of 36 family members of 3 generations was constructed. Chromosome analysis was carried out in G- and C-banding from peripheral blood cultures of 19 family members. Distinct heteromorphism in the chromosome 1qh region was detected in 15 (79%) of them, including all 3 cancer patients investigated.