UCN-01 dose-dependent inhibition of normal hyperproliferative cells in mice.

UCN-01 is a hydroxylated derivative of staurosporine and a potent protein kinase C (PKC) inhibitor. Interest in the potential usefulness of this compound as an anticancer drug stems mainly from its unique anti-signaling, growth-arresting properties on tumor cells. This include activation of CDC2 kinase (CDK1) which interacts with either cyclin A or cyclin B1 at the G1 or G2/M border, suggeting that this event is one of the major consequences of the drug action on eukaryotic cells. Nonetheless, the antiproliferative activity of UCN-01 on normal rapidly dividing cells (intestinal epithelial and bone marrow cells) is not well documented. Thus, the main objective of this study was to investigate the in vivo antiproliferative activity of UCN-01 on these normal hyperproliferative cells and evaluate whether cellular response to UCN-01 could be modulated in the presence of DNA damage. Mice were injected i.m. with a single dose of UCN-01 (2.5 mg/kg-20 mg/kg) followed 3 and 24 h later by in vivo BrdU labeling for 1 h. At autopsy, bone marrow cells were collected and fixed for dual parameter BrdU/DNA flow cytometry. Different regions of the gut were also fixed for immunoperoxidase BrdU assays. Newly replicated cells were mainly located in the lower compartments of the crypt columns and were scored for BrdU stained nuclei using an image analysis system. A comparison between groups showed that 5 mg/kg UCN-01 induced inhibition in BrdU incorporation at 3 and 24 h, as compared to the other groups injected with various doses of UCN-01. Flow cytometric analysis of bone marrow cells stained with fluorescein tagged anti-BrdU (FITC) along with propidium iodide (PI) also showed inhibition in BrdU incorporation of S phase fraction cells in mice treated with 5 mg/kg UCN-01. These bone marrow cells were arrested primarily in the G1 phase of the cell cycle. The colony-forming unit (CFU) assay of the bone marrow cells was then used to determine the level of drug interaction of UCN-01 and, topotecan, a topoisomerase I inhibitor, at a fixed dose ratio. An antagonistic drug interaction (CI > 1) was observed as determined by the median-effect analysis. However, an additive interaction (CI = 1) was obtained with the use of camptothecin or 10,11-methylenedioxycamptothecin and UCN-01. The results of the in vitro drug interaction with UCN-01 may predict protection from topotecan-induced bone marrow toxicity.

Abrogation of cell cycle checkpoint controls during malignant transformation of syrian hamster embryo cells is associated with decreased sensitivity to apoptosis.

Malachite green (MG), consisting of green crystals with a metallic luster, is highly soluble in water, cytotoxic to various mammalian cells, and may act as a liver tumor promoter. In view of its industrial importance and possible exposure to human beings, MG poses a potential environmental health hazard. We have reported earlier the malignant transformation of Syrian hamster embryo (SHE) cells by MG. In this study, we investigated the effects of MG on flow cytometric cell cycle phase distribution of normal and MG-transformed SHE cells in asynchronous and synchronous cell populations. DNA flow cytometric analysis indicated that culturing cells for 48 hours in a medium containing MG 0.1 microg/mL induced G2/M arrest in normal cells. Malignant-transformed cells showed no such accumulation of cells at the G2/M phase of the cell cycle in response to MG. Synchronization studies indicated that in the control, both in the presence and absence of MG, cells followed a normal cell cycle pattern up to 16 hours. After 16 hours, in the absence of MG, cells continued a normal cell cycle, whereas in the presence of MG they accumulated at the G2/M phase of the cell cycle. This pattern of accumulation of cells at the G2/M checkpoint control was not observed in either untreated or MG-treated transformed cells. We also studied the effects of MG on the induction of apoptosis using flow cytometric FSC/SSC scatter plots in normal and transformed SHE cells. Flow cytometric analysis showed a dose- and time-dependent induction of apoptosis by MG in control cells, whereas induction of apoptosis by MG was marginal in transformed cells. In the present study, we demonstrated the efficient operation of the G2/M checkpoint control, apoptosis in control SHE cells, the abrogation of checkpoint controls, and decreased sensitivity to apoptosis in transformed SHE cells.

Genes in the first and fourth inversions of the mouse t complex synergistically mediate sperm capacitation and interactions with the oocyte.

The t haplotypes (t) are recent evolutionary derivatives of an alternate form of the mouse t complex region located at the proximal end of chromosome 17. This variant form of approximately 1% of the mouse genome is a source of mutations altering numerous sperm functions crucial for fertilization. Males that carry two t haplotypes (t/t) are invariably sterile. t haplotypes contain four inversions relative to the wild-type t complex (+), so that in matings involving a +/t heterozygote, t is usually transmitted as a single unit. However, rare recombinants have been recovered, which carry only part of the t genotype and express only some of the t-dependent phenotypes. Use of these partial t haplotypes in genetic crosses has resulted in the general location of the two major t male sterility factors, S1 and S2, within inversions 1 and 4, respectively. Since sterility can result from a plethora of sperm defects, we have made a detailed study of various functional parameters of sperm from mice carrying S1 or S2 heterozygously or homozygously or in combination. Both S1 and S2 contain mutations altering sperm functions, including motility, capacitation, binding to the zona pellucida, binding to the oocyte membrane, and penetration of the zona pellucida-free oocyte. Therefore it seems clear that each of these factors contains multiple genes contributing to sterility. Furthermore, our results indicate that genes within S1 interact with genes in S2 for all sperm functions examined. However, S1 and S2 genes affecting motility interact in a purely additive fashion, while S1 and S2 genes affecting most other sperm characteristics interact in a synergistic manner. Additionally, the patterns of synergism between S1 and S2 for abnormalities in capacitation, sperm-oolemma binding, and zona-free oocyte penetration are nearly identical. This suggests that these three defects are caused by mutation of the same gene within each sterility factor. These findings will not only be instrumental in matching the various t haplotype sperm defects to candidate genes for S1 and S2, but will facilitate a more comprehensive understanding of the cellular and genetic mechanisms underlying t haplotype male sterility.

Malachite green induced malignant transformation of Syrian hamster embryo (SHE) cells in primary culture: transformation is associated with enhanced expression of altered p53, bcl-2 and decreased sensitivity to apoptosis.

Malachite green (MG) consisting of green crystals with a metallic lustre, is highly soluble in water, cytotoxic to various mammalian cells and also acts as a liver tumor promoter. In view of its industrial importance and possible exposure to human beings, MG poses a potential environmental health hazard. We have previously reported the malignant transformation of Syrian hamster embryo (SHE) cells by MG. In this study, we have made an attempt to study the effects of MG on the induction of apoptosis in normal and MG transformed Syrian hamster embryo cells and the expression of altered p53 and bcl-2 immunohistochemically. Induction of apoptosis was detected by flow cytometry on the basis of G0/G1 hypodiploid peak, Tunel assay and DNA ladder pattern. Flow cytometric analysis showed a dose and time dependent induction of apoptosis by MG in control cells whereas induction of apoptosis by MG was marginal in transformed cells. Tunel assay and DNA ladder pattern also showed decreased apoptosis in transformed cells by MG compared to controls. Immunostaining studies showed intense staining for p53 in transformed cells whereas no staining was observed in control cells. Also transformed cells showed overexpression of bcl-2 with exclusive nuclear localization compared to controls which did not show staining. The present study indicated that MG transformed Syrian hamster embryo cells have decreased sensitivity to apoptosis compared to normal cells and overexpression of altered p53 and bcl-2 seems to be conferring resistance to MG induced apoptosis.

An improved mouse sperm-oocyte plasmalemma binding assay: studies on characteristics of sperm binding in medium with or without glucose.

Sperm binding to the oocyte plasmalemma is crucial to subsequent steps in fertilization. However, the usual in vitro assay for sperm-oocyte binding does not distinguish between nonspecific attachment and specific binding leading to fusion and penetration. Since zona pellucida-free pronuclear zygotes should not bind sperm (because of the block to polyspermy at the level of the oocyte membrane), a procedure has been developed to remove virtually all sperm from zona-free pronuclear zygotes (2PNZ). After six washes with a 90-microm-bore pipette, there were 0.5 +/- 0.2 sperm/2PNZ (n = 83). Therefore, these washing conditions were used to define sperm-oocyte binding. The relationship of binding to oocyte penetration was determined for outbred mouse oocytes coincubated in complete medium with (B6 x 129)F1 hybrid sperm (10(7)/ml). Binding was maximal (29 +/- 5 sperm per oocyte) during the first 30 minutes but decreased significantly to 4.6 +/- 1.4 by 60 minutes of coincubation (over 10 trials). Oocyte penetration was 99 +/- 1% by 30 minutes, while the number of decondensed sperm nuclei per oocyte increased significantly to 7.5 +/- 0.6 at 60 minutes. These data suggest that the block to polyspermy involves detachment of bound sperm. Similar coincubations were carried out in medium without glucose (NoG), as this medium has been reported to inhibit fusion without affecting binding. However, binding was only 11 +/- 2 at 30 minutes but increased to 25 +/- 4 at 60 minutes, suggesting that binding was retarded in NoG. When gametes were coincubated in NoG for 5 hours, about half of the oocytes were penetrated, suggesting that the lack of glucose did not inhibit fusion but instead delayed it. These data suggest that if sperm binding is to be determined in complete medium, the time of the block to polyspermy should be determined prior to selecting the appropriate time to assay binding. Furthermore, using the same coincubation period for the binding assay in control and treated sperm may not be appropriate if the treatment alters the time of maximal binding.

Expression of Fas and Fas ligand in Hodgkin's disease.

Fas and Fas ligand expression were investigated in twenty two cases of classical Hodgkin's disease (HD) by immunohistochemistry. While Reed-Sternberg (RS) cells in 7/22 (32%) cases expressed Fas ligand, reactive lymphoid cells expressed Fas ligand in only 2 (9%) cases. In 20/22 (91%) cases, the RS cells expressed Fas. A higher proportion of RS cells in the nodular sclerosis subtype expressed Fas as compared to the mixed cellularity subtype. In 18/22 (82%) cases, Fas expression was also noted in the reactive lymphoid cells. In eight cases, the reactive lymphoid cells were also analyzed by flow cytometry and a majority of them were CD4+CD45RO+. Most of these activated T-cells expressed Fas but were negative for Fas Ligand. To investigate the co-expression of Fas and Fas Ligand in the RS cells, six cases were subjected to Fas and Fas ligand immunostaining on consecutive sections. The co-expression was documented in the RS cells in four of six cases. These six cases with expression of both Fas and Fas ligand were investigated for the incidence of apoptosis. There was no statistically significant relationship between expression of Fas on reactive cells, expression of FasL on RS cells and the proportion of apoptotic reactive cells. In all these cases apoptosis was not observed in the RS cells. Thus Fas - FasL interactions may not lead to apoptosis of the RS cells.

Flow cytometric analysis of estrogen, progesterone receptor expression and DNA content in formalin-fixed, paraffin-embedded human breast tumors.

Flow cytometric analysis of estrogen (ER) and progesterone (PgR) receptor expression in archival human breast tumors is relatively difficult. We have used enzyme digestion and microwave antigen retrieval procedures for multiparametric flow cytometric analysis of ER and PgR expression and DNA content in nuclei isolated from formalin-fixed/paraffin-embedded primary breast tumors. Deparaffinized rehydrated tissue sections treated with pepsin were subjected to microwave irradiation for unmasking of ER and PgR antigenic sites. Biotinylated ER antibody and streptavidin-fluorescein isothiocyanate (FITC) were used for ER labeling and PgR antibody with phycoerythrin labeled goat anti-mouse antibody was used for PgR labeling. Counter staining with propidium iodide-RNase was used for determination of cellular DNA content. Our results show that enzyme digestion and microwave treatment of formalin-fixed, paraffin-embedded breast tumors can be successfully used for the multiparametric analysis of nuclear hormone receptor expression and DNA content by flow cytometry.

Malignant transformation of Syrian hamster embryo (SHE) cells in culture by malachite green: an agent of environmental importance.

Malachite green (MG), consisting of green crystals with a metallic lustre, is very soluble in water and is highly cytotoxic to mammalian cells in culture and also acts as a liver tumour promoter. In view of its industrial importance and possible exposure to human beings, MG poses a potential environmental health hazard. Accordingly, we have studied the effect of MG on the formation of free radicals using Electron Spin Resonance (ESR) analysis with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trapping agent. ESR analysis showed formation of reactive free radicals during exposure of MG to Syrian hamster embryo (SHE) cells. As per mechanism-based toxicology in cancer risk assessment, the chemicals that have the potential to be metabolized to active free radical species could be human cancer hazards. So, we have investigated the effect of MG on the formation of Type II and Type III morphologically transformed foci using SHE cell transformation assay. MG induced dose related transformed foci. Some of these transformed foci were taken out using selective trypsinisation and established immortal cell lines. One of these immortal cell lines was characterized extensively. This immortal cell line showed enhanced DNA synthesis in the form of BrdU incorporation, increased presence of proliferating cell nuclear antigen (PCNA), bcl-2 and p53 proteins by immunohistochemistry. When these immortal cells were injected subcutaneously into nude mice, they developed tumors which were transplantable and histopathologically sarcomas. The present studies indicate that MG could be a potential candidate for two year chemical carcinogenesis rodent bioassays.

CD40-ligation-mediated protection from apoptosis of a Fas-sensitive Hodgkin's-disease-derived cell line.

Modulation of Fas expression and function by CD40 ligation was investigated in the Fas-sensitive human Hodgkin's disease cell line HDLM2. The recombinant human trimeric soluble CD40L (sCD40L) protected this cell line from apoptosis induced by an agonistic Fas antibody at all concentrations tested. sCD40L also protected HDLM2 when added up to 2 h after Fas ligation. Apoptosis induced by a cell-permeable synthetic ceramide could not be prevented by sCD40L. Thus, CD40 ligation is likely to intervene in the early phases of the Fas signal transduction pathway. When CD40 ligation preceded Fas ligation, it rendered the cells refractory to Fas-induced apoptosis. sCD40L-mediated protection could not be attributed to reduction in surface Fas expression, increase in Bcl-2 levels or to increase in the levels of soluble Fas isoforms.

High-resolution mapping of sperm function defects in the t complex fourth inversion.

Structural variants of the mouse Chr 17-specific t complex, known as t haplotypes, express factors that alter the ability of sperm to carry out their roles in the normal fertilization process. In previous studies of males carrying heterospecific combinations of the t complex, we discovered a unique M. spretus/t haplotype phenotype of male sterility. In additional studies with mice carrying a series of M. spretus-M. m. domesticus recombinant Chr 17 homologs and a complete t haplotype (S-+/t), we monitored physiological aspects of sperm function to map a locus (Hst6) responsible for expression of the t-specific "curlicue" sperm flagellar curvature phenotype to 1 cM within the fourth inversion of the t complex. In the present report, we quantitatively analyze the in vitro capability of sperm from mice with similar S-+/t Chr 17 genotypes to fertilize zona pellucida-free mouse eggs. The results identify a locus, Stop1, mapping distal to Pim1, with acute effects on the ability of sperm to penetrate the oolemma. The data suggest that Stop1 is a complex locus consisting of at least two genetic elements, a proximal one overlapping the Hst6 locus, and another, distal to the Hst6 locus. Further quantitative analyses of the "curlicue" phenotype produced by sperm derived from these same animals indicate that expression of this chronic flagellar curvature phenotype also derives from at least two elements, both mapping within the Hst6 locus. Thus, these studies provide higher resolution mapping of the molecular basis of t haplotype-specific sperm dysfunction emanating from In(17)4.

Malignant transformation of Syrian hamster embryo (SHE) cells in primary culture by malachite green: transformation is associated with abrogation of G2/M checkpoint control.

Malachite green (MG), consisting of green crystals with a metallic lustre, is highly soluble in water, cytotoxic to various mammalian cells and also acts as a liver tumour promoter. In view of its industrial importance and possible exposure to human beings, MG poses a potential environmental health hazard. We have earlier reported the malignant transformation of Syrian hamster embryo (SHE) cells by MG. In this study, we have studied the effects of MG on cell cycle phase distribution of normal and MG transformed Syrian hamster embryo cells in asynchronous and synchronous cell population. DNA flow cytometric analysis indicated that culturing cells for 48 h in medium containing MG at different concentrations induced dose-dependent G2/M arrest in normal cells. Malignantly transformed cells showed no such dose-responsive accumulation of cells at the G2/M phase of the cell cycle in response to MG. Synchronization studies indicated that in the control, both in the presence and absence of MG, cells followed a normal cell cycle pattern up to 16 h. After 16 h in the absence of MG, cells continued a normal cell cycle, whereas in the presence of MG they accumulated at G2/M phase of the cell cycle. This pattern of accumulation of cells at the G2/M checkpoint control was not observed in either untreated or MG-treated transformed cells. The present study indicates efficient operation of G2/M checkpoint control in control SHE cells and its abrogation in transformed SHE cells.

Prognosis of breast cancer: evidence for interaction between c-erbB-2 overexpression and number of involved axillary lymph nodes.

The prognostic significance of c-erbB-2 oncogene amplification or overexpression in relation to axillary lymph node metastasis is controversial. We investigated this question in 159 cases of operable breast cancer: 56 patients with node negative disease and 103 patients with pathological involvement of axillary lymph nodes. c-erbB-2 overexpression was assessed by immunohistochemistry using a polyclonal antibody raised against a synthetic peptide fragment of the oncoprotein. The overall incidence of c-erbB-2 overexpression was 35%. c-erbB-2 overexpression was significantly related to survival when all patients were considered (P = 0.0124), and also for patients with positive axillary lymph nodes (P = 0.0026). c-erbB-2 overexpression had no influence on survival of node negative patients (P = 0.7972). A multivariate survival analysis using the Cox proportional hazard model revealed that number of involved lymph nodes, c-erbB-2 overexpression, ER status, and tumour size were independently related to prognosis (P = 0.0000, 0.0012, 0.0112, and 0.0204, respectively). When an interaction term was introduced in the Cox model between c-erbB-2 overexpression and number of involved axillary lymph nodes, a statistically highly significant interaction between these two factors was observed (P = 0.0002), suggesting that the expression of prognostic power of c-erbB-2 overactivity is related to the number of involved axillary lymph nodes. The 159 patients were then subdivided into three groups: node negative (-ve) (56); 1-6 node positive (+ve) (55); and > or = 7 node +ve (48). This cutoff criterion gave the most numerically equitable distribution of the 159 patients into three groups. The relative risk of death increased stepwise from 0.86 (95% CI 0.26-2.78) for node negative patients, to 1.95 (95% CI 0.82-4.63) for 1-6 node positive patients, to 2.23 (95% CI 1.15-4.35) for > 7 node positive patients. Our results suggest that the prognostic influence of c-erbB-2 overexpression increases arithmatically with increasing number of involved axillary lymph nodes.

Oestrogen and progesterone receptor estimation by enzyme-immunoassay on tissues removed before and after a modified radical mastectomy.

To investigate the possibility of receptor degradation due to devascularization of the tumour during mastectomy, oestrogen and progesterone receptors (ER and PgR) were measured by an enzyme-immunoassay (EIA) in 59 cases of primary breast cancer on samples taken before and after performing a modified radical mastectomy. Pre- and post-mastectomy samples from the same patient were analysed simultaneously in the same assay run. There was 86.4% and 93.2% agreement respectively in ER and PgR status between samples removed before and after surgery. When actual values were analysed, the post-mastectomy values were higher or lower than pre-mastectomy values with similar frequency. These random variations could be attributed to heterogeneous distribution of receptors within a tumour. The overall correlation between pre- and post-mastectomy values was excellent (ER: r = 0.810, P < 0.001; PgR: r = 0.706, P < 0.001). Devascularization of the tumour during surgery does not seem to affect the integrity of the epitopes recognized by monoclonal antibodies against ER and PgR to any significant extent.

DNA analysis of breast cancer by flow cytometry & correlation with other prognostic parameters.

Flow cytometric estimation of DNA content (ploidy and S-phase fraction--SpF) was done on breast cancer tissues from 171 patients. Twenty eight per cent of the tumours were diploid and 72 per cent were aneuploid. SpF was measurable in 82 DNA histograms; of these 22.4 per cent had SpF less than 10 per cent, 34.1 per cent had SpF between 10-20 and 43.5 per cent had SpF greater than 20 per cent. The mean SpF of the measurable histograms was 19.01 per cent with a range 1.78 to 45.19 per cent. A significant correlation between DNA ploidy and SpF was observed (P less than 0.01). Eighty nine per cent of diploid tumours had SpF less than 10 per cent and 73 per cent of aneuploid tumours had SpF greater than 20 per cent. A significant correlation was also found between ploidy and SpF and oestrogen receptor (ER) status of the tumours (P less than 0.05) and between SpF and progesterone receptor (PgR) status of the tumours (P less than 0.05), but not between ploidy and PgR status of the tumours. A significant direct correlation was observed between SpF and tumour grade (P less than 0.05), but not between ploidy and tumour grade. No correlation was observed between DNA ploidy and SpF and tumour type, tumour size, axillary lymph node involvement, age and menopausal status of the patients. Although the incidence of breast cancer is one-third of that reported in the Western countries, there is apparently no biological difference between the various parameters studied.

Estrogen & progesterone receptors measurement in breast cancer with enzyme-immunoassay & correlation with other prognostic factors.

Estrogen and progesterone receptor (ER and PgR) estimation was carried out by an enzyme-immunoassay by a 'sandwich' technique using two different monoclonal antibodies against each receptor on 508 consecutive breast cancer samples. 43.9 per cent of the tumours were ER+ve and 26.6 per cent were PgR+ve; 23.8 per cent were both ER and PgR+ve, 53.3 per cent were both ER and PgR-ve, 20.0 per cent were ER+ve PgR-ve and 2.8 per cent were ER-ve PgR+ve. Both ER and PgR positivity was associated with increasing age, and this was seen within both pre and post-menopausal subgroups. Grades I and II tumours were more often ER and PgR+ve compared with grade III tumours, indicating that receptor positivity is a marker of a more well differentiated tumour phenotype. Receptor positivity was higher in primary tumours compared to that in metastatic tissues. The proportion of tumours that was ER+ve was found to vary among the four major religious communities, viz., Hindu, Muslim, Christian and Parsi, and this variation was significant in the overall analysis (P less than 0.01). The Christians had the highest rate of ER+ve tumours while the Muslims had the lowest rate. No correlation was observed between ER and PgR status and axillary nodal involvement or tumour size, suggesting that ER and PgR are independent prognostic factors in breast cancer. We found the EIA method to be an easy and rapid technique for ER and PgR analysis and which requires a small amount of tissue and does not involve the use of radioisotopes.

Correlations between c-erb beta-2 oncogene amplification and the expression of its mRNA and protein in human breast carcinomas.

Nineteen human breast tumours were analysed for c-erb beta-2 gene amplification, the over-expression of its mRNA and the production of its protein residue by southern blot, northern blot and western blot technique respectively. Protein expression was also assessed by immunohistochemistry on paraffin sections, c-erb beta-2 gene amplification was found in 6 of 19 (31%) tumours. Over-expression of c-erb beta-2 mRNA was found in 7 of 19 (37%) tumours. Expression of the 185 kd c-erb beta-2 protein product was detected in 3 of 19 (16%) tumours by western blotting and in 5 of 19 (26%) by immunohistochemistry. In only 4 tumours with an amplified c-erb beta-2 gene was there a clear association between all three parameters. In view of the conflicting reports in the literature concerning c-erb beta-2 gene amplification or protein over-expression (assessed by western blot or immunohistochemistry) and prognosis of breast cancer, studies in which these parameters are correlated individually with prognosis in the same group of patients are needed.

Differences in oestrogen receptor status and histological grade of breast cancer in different religious communities in Bombay.

The various religious communities in India viz.Hindu, Muslim, Christian, Parsi have different breast cancer incidence rate. It is not known whether there might also exist differences in biological properties of breast cancer between these communities. To investigate this possibility we have studied the distribution of oestrogen receptor (ER) status and histological grade of tumour in these four communities. Significant differences were observed in the overall distribution of ER positivity and histological grade between the communities P less than for both parameters). Christians had the highest incidence of ER +ve (65.2%) and grade I + II tumours (16.0%), while Muslims had the lowest incidence of ER +ve (35.8%) and Grade I + II tumours (4.7%). In general, we found a significant positive relationship between ER status and age of the patient (p less than 0.0.1). The mean age of the christians was slightly but significantly higher than that of the Hindus and Muslim. The difference ER positivity between the communities might, therefore, be partly (but probably not wholly) explained by difference in age of the patients. However, the difference with respect to grade of tumour cannot be explained as a function of age since no significant association was found between grade of the tumour and age of the patient. Further investigation with respect to difference in the biology of the breast cancer between the communities are warranted.

A comparative study of dextran coated charcoal (DCC) and enzyme immunoassay (EIA) methods for oestrogen receptor (ER) estimation in breast cancer.

Oestrogen receptor (ER) concentrations were measured in 100 breast cancer cytosols using both the dextran coated charcoal assay (DCC with 6--point scatchard plot) and a newly developed enzyme linked immunoassay (EIA) using a monoclonal antibody against ER. The efficiency of the two methods was compared. A highly significant correlation was observed between the ER levels determined by DCC and EIA methods (r = 0.94, p less than 0.001). The mean ER-EIA value (43.25 +/- 74.77) was, however, significantly higher than the mean ER-DCC value (18.00 + 37.27) (p less than 0.001); in both pre- menopausal (p less than 0.001) and post-menopausal (p less than 0.001) groups. Using a cut-off point at 10 fmo/mg protein for ER-EIA and 3 fmol/mg protein for ER-DCC/to distinguish ER +ve and -ve tumours, a 97% concordance between the two assays was achieved. The EIA method appears to be preferable to the DCC method because it is easy to perform, rapid, requires less tissue and does not involve the use of radioactive substances.