Progestins inhibit the growth of MDA-MB-231 cells transfected with progesterone receptor complementary DNA.

Because progesterone exerts its effects mainly via estrogen-dependent progesterone receptor (PgR), the expression of progesterone's effects may be overshadowed by the priming effect of estrogen. PgR expression vectors were transfected into estrogen receptor (ER)-alpha and PgR-negative breast cancer cells MDA-MB-231; thus the functions of progesterone could be studied independent of estrogens and ERs. Eight stable transfectant clones expressing both PgR isoform A and B were studied for their growth response to progesterone and its analogues. Although progesterone had no effect on growth in the control transfectant, the hormone markedly inhibited DNA synthesis and cell growth in all of the PgR-transfectants dose-dependently from 10(-12)-10(-6) M. This growth inhibition was associated with an arrest of cells in the G0/G1 phase of the cell cycle. Progestins medroxyprogesterone acetate, Org2058, and R5020 also strongly inhibited DNA synthesis, and their doses required for maximal inhibition of 60-70% were 10(-17) M, 10(-13) M, and 10(-7) M, respectively. Antiprogestin ZK98299 alone had no effect, but the compound was capable of counteracting the inhibitory effect of progesterone. In contrast, RU486 inhibited DNA synthesis, and it showed no further effects when it was used concurrently with progesterone. These results indicate that progestins are per se antiproliferative via a PgR-mediated mechanism in breast cancer cells. More importantly, we have shown that progestins may exert effective inhibitory control over the cell growth if the PgR expression is reactivated in ER- and PgR-negative breast cancer cells.

Progesterone induces focal adhesion in breast cancer cells MDA-MB-231 transfected with progesterone receptor complementary DNA.

Since the effects of progesterone are mediated mainly via estrogen-dependent progesterone receptor (PR), the expression of the effects of progesterone may be masked or overridden by the influence of estrogen under conditions in which priming with estrogens is required. We have established a PR-positive but estrogen receptor-alpha (ER-alpha) negative breast cancer cell model by transfecting PR cDNA into ER-alpha- and PR-negative MDA-MB-231 cells in order that the functions of progesterone can be studied independently of estrogens. We have demonstrated using this model that progesterone markedly inhibited cell growth. We have also discovered that progesterone induced remarkable changes in cell morphology and specific adhesion structures. Progesterone-treated cells became considerably more flattened and well spread than vehicle-treated control cells. This was associated with a striking increase of stress fibers, both in number and diameter, and increased focal contacts as shown by the staining of focal adhesion proteins paxillin and talin. There were also distinct increases in tyrosine phosphorylation of focal adhesion protein paxillin and focal adhesion kinase in association with increased focal adhesion. The staining of tyrosine-phosphorylated proteins was concentrated at focal adhesions in progesterone-treated cells. More interestingly, monoclonal antibody (Ab) to beta1 integrin was able to inhibit progesterone-induced cell spreading and formation of actin cytoskeleton. To our knowledge, this is the first report describing a direct effect of progesterone in inducing spreading and adhesion of breast cancer cells, and beta1-integrin appeared to play an essential role in the effect. It is known that the initial step of tumor metastasis is the breakaway of tumor cells from primary tumor mass when they lose the ability to attach. Hence, progesterone-induced cell spreading and adhesion may have significant implications in tumor metastasis.

Vinorelbine induces apoptosis and caspase-3 (CPP32) expression in leukemia and lymphoma cells: a comparison with vincristine.

Vinorelbine (NVB) is a novel vinca alkaloid FDA approved for use in some advanced carcinomas. However, its role in non-Hodgkin's lymphoma (NHL) is still not well defined. NVB is an antimicrotubule agent, but as yet, it is not known whether it induces apoptosis. By flow cytometry using nuclear staining (propidium iodide) and annexin V, we demonstrated that NVB and vincristine (VCR) induced both mitotic arrest and apoptosis in leukemia and lymphoma cells, in a drug exposure time dependent manner. Cell cycle kinetics in 3 different cell lines varied during vinca alkaloid treatment. The annexin V method showed that apoptosis, as opposed to necrosis, was the dominant mode of cell kill of chemosensitive leukemia and lymphoma cells. Phosphatidylserine expression on the cell surface was detectable as a hallmark of apoptosis at earlier drug exposure when compared to conventional flow cytometry with PI staining. By Western blot analysis, we demonstrated that CPP32 or caspase-3, a critical apoptosis inducer, and its active subunits p20 and p11 were upregulated in chemo- and apoptosis-sensitive lymphoma and leukemia cells treated with NVB. Our data contributes to the emerging hypothesis suggesting that widely divergent exogenous stimuli and chemotherapeutic agents can effect apoptosis in cancer cells via different pathways involving the caspases. We believe that vinorelbine may be a potentially important drug in the treatment of NHL in the future.

The radiometric assay of alkaline phosphatase activity with 125-I-labelled phenolphthalein monophosphate.

The coupling of 125-I to phenolphthalein monophosphate is described. Hydrolytic cleavage of the phosphate group by alkaline phosphatases (EC 3.1.-3.1) releases labelled phenolphthalein which is extracted into ethyl acetate at pH 10. This forms the basis of a sensitive assay for the enzyme with the advantages of sensitivity and rapidity from the use of a gamma-emitter. Results with Escherichia coli and calf intestine alkaline phosphatases are presented.

The radiometric assay of microsomal UDP-glucuronyltransferase using 125I-labelled phenolphthalein.

A rapid and sensitive method is described for assaying UDP-glucuronyl-transferase (EC 2.4.1.17) activity. The substrate is the relatively apolar phenolphthalein, labelled with 125I on one of its two phenolic rings leaving a free hydroxyl group for glucuronidation. Extraction with ethyl acetate leaves the glucuronide in the aqueous reaction medium which is then counted. Less than 10 mug microsomal protein is required and the glucuronidation of 10 ng 125I-labelled phenolphthalein (0.03 nmoles) is easily detected.

Graves' ophthalmopathy in the absence of elevated free thyroxine and triiodothyronine levels: prevalence, natural history, and thyrotropin receptor antibody levels.

The aims of this study were to (a) determine the prevalence of patients without elevated thyroid hormone levels in Graves' ophthalmopathy (GO) using current generation free thyroid hormone assays, (b) measure the prevalence of thyrotropin receptor antibodies (TRAb) in these cases, and (c) identify possible predictors of hyperthyroidism. Over a 30-month period, 1020 cases of thyroid eye disease were evaluated, of which only 19 (1.9%) met the diagnostic criteria. Ten (1%) had subclinical thyrotoxicosis, 7 (0.7%) were euthyroid, and 2 (0.2%) were hypothyroid as determined by a third-generation thyrotropin (TSH) assay. TRAb levels were measured in 16 of these 19 patients. The prevalence of TRAb varied according to the assay used. Polyethylene glycol-extracted thyroid-stimulating immunoglobulin (PEG-TSI), unfractionated thyroid-stimulating immunoglobulin (uTSI), first-generation porcine TSH-binding inhibitory immunoglobulin (pTBII), and second-generation human TSH-binding inhibitory immunoglobulin (hTBII) assays were positive in 93.8%, 50%, 18.8%, and 81.3% of patients, respectively. TRAb was detected by at least one method in all patients. Patients were followed up for 15 to 45 months. Hyperthyroidism developed in 4 patients (25%). Suppressed TSH levels and elevated TBII were predictors of hyperthyroidism. When sensitive assays are used, the prevalence of GO patients without elevated thyroid hormone levels is extremely low. The sensitivities of assays for TRAb detection differ substantially in these cases. PEG extraction improves the detection rate of TSI (p = 0.02), and hTBII assays improve the detection of TBII in these patients (p = 0.002). The high prevalence of TRAb in such cases supports a role for these antibodies in the pathogenesis of thyroid-associated eye disease.

The combination of absent thyroid peroxidase antibodies and high thyroid-stimulating immunoglobulin levels in Graves' disease identifies a group at markedly increased risk of ophthalmopathy.

Among Graves' Disease (GD) patients, we have observed an unexpectedly high prevalence of antithyroperoxidase antibody (TPOAb) and antithyroglobulin antibody (TgAb) negativity in those with severe ophthalmopathy. To study the possible role of thyroid autoantibodies in the pathogenesis of Graves' ophthalmopathy (GO), TPOAb, TgAb, thyroid-stimulating immunoglobulin (TSI), and thyrotropin-binding inhibitory immunoglobulin (TBII) levels were measured, and the presence or absence of GO was assessed by a single observer in 100 consecutive patients with newly diagnosed, untreated GD who were nonsmokers. Ophthalmopathy was present in 43 patients. TSI levels (p = 0.001), and the prevalence of TPOAb-negativity (p = 0.002) were significantly higher in patients with ophthalmopathy compared to those without. Logistic regression analysis showed that TSI levels (p = 0.005) and the absence of TPOAb (p = 0.0025) were independent predictors of GO. No correlation between TBII or TgAb and eye disease was found. The prevalence of GO increased with each quartile of TSI levels. The prevalence was 20%, 36%, 52%, and 64% in the first, second, third and fourth quartiles of TSI, respectively. The odds ratio of GO (with 95% confidence intervals) when TSI levels were above the median level (1640%) was 3.6 (1.5-8.0), when TPOAb was negative it was 5.0 (1.7-14.4), and with both risk factors it was 36.6 (4.3-313.5). The prevalence of ophthalmopathy in this last group was 92.9%. The combination of negative TPOAb and high TSI levels appears to be associated with a markedly increased risk of clinically evident ophthalmopathy.

99Tcm-polyclonal IgG and 99Tcm nanocolloid scans in orthopaedics: a comparison with conventional bone scan.

The bone scan is sensitive in detection of active bone/joint lesions. A normal bone scan virtually excludes the presence of an inflammatory process with high precision, but the poor specificity of bone scans is well known. In recent years, various new agents including 99Tcm-hexamethylpropylene amine oxime (HMPAO)-labelled white blood cells, nanocolloid, polyclonal IgG, anti-granulocyte antibody, 111In-labelled IgG, leucocytes, chemotactic peptides etc. have been widely evaluated in inflammatory imaging, especially in the orthopaedic context. This study was undertaken to compare the usefulness of 99Tcm-nanocolloid and 99Tcm-polyclonal IgG in the detection of focal bone/joint inflammation. Twenty-seven patients with a common presentation of bone/joint pain resulting from various pathologies were included in the study. A total of 47 lesions were imaged. The overall sensitivity and specificity of both nanocolloid scan and IgG scan were identical with 95% sensitivity and 100% specificity, in detecting inflammatory foci. However, specificity dropped to 18% with nanocolloid scans and 16% with IgG scans when an attempt was made to distinguish noninfective from infective inflammatory processes; thus neither type of scan permits differentiation between septic and nonseptic inflammatory processes with sufficient accuracy. As both nanocolloid and IgG scans are equally sensitive and specific in detecting inflammation, the choice of type of scan will depend on cost, imaging time and availability of the radiopharmaceutical.

A non-invasive isotope dilution technique for quantifying hepatic blood flow using radiolabelled red blood cells.

Clinically significant changes in hepatic haemodynamics accompany the development of portal hypertension, hepatocellular carcinoma, liver metastases and liver cirrhoses, and after major liver resection. Hepatic blood flow parameters, such as hepatic arterial flow (HAF), hepatic portal flow (HPF), total hepatic blood flow (THBF) and hepatic perfusion index (HPI), are useful adjuncts to the diagnosis of liver pathology, the evaluation of disease progress and prognostication. Here, we describe a non-invasive method that combines the measurement of these parameters in a single study in real time. Red blood cells from eight pigs were labelled with 99Tc(m) using an in-vitro method and re-injected into the pigs. Data acquisition over the heart, lungs, liver and kidneys was started immediately and a blood sample was obtained 15 min post-injection. Hepatic arterial flow was determined from the ratio of the maximum gradients between the integrated time-activity curve of the left ventricle and the first-pass time-activity curve of the liver before the peak of the kidneys time-activity curve. The hepatic perfusion index was determined by comparing the slope of the liver time-activity curve before and after the kidney peak. Hepatic portal flow was determined from the hepatic arterial flow and the hepatic perfusion index, and total hepatic blood flow was determined as the sum of arterial and portal flow. The results were compared against those obtained from a clearance method using 99Tc(m)-DISIDA. The average hepatic perfusion index was 0.38, and the average hepatic arterial flow and hepatic portal flow were 168.3 +/- 52.9 and 274.6 +/- 60.1 ml x min(-1) respectively. The average total hepatic blood flow was 442.8 +/- 53.5 ml x min(-1), while the total hepatic flow determined by 99Tc(m)-DISIDA clearance was 419.7 +/- 62.6 ml x min(-1). No significant difference in total hepatic blood flow was found between the two methods. The results of this study show that it is possible to obtain all hepatic haemodynamics data in a single study using a non-invasive method.

Differential oxygen sensitivities in G6PDH activities of cultured keloid and normal skin dermis single cells.

Cell cultures of excised keloids and biopsied normal skin were established from patients of the Plastic Surgery Department of Singapore General Hospital and their single cells quantitated for glucose-6-phosphate dehydrogenase (G6PDH) activity using microspectrophometry. G6PDH has been cited as a transformation-linked discriminant. Analysis of variance shows no difference in overall activity between skin and keloid cells, but under oxygen saturation conditions, keloid G6PDH activity was significantly higher than skin G6PDH activity, although they were almost identical under nitrogen saturation conditions. Differential oxygen sensitivity in G6PDH activities between malignant and non-malignant cells have been reported, but its occurrence between keloid and normal skin cells is novel, especially as the keloid is regarded as a benign tumor with a zero carcinogenicity rate.

Autoimmune disease--pathogenesis through molecular mimicry at the tripeptide level.

Increasingly it is being discovered that short segments of proteins can provoke an immune response. Sequential determinants are as important as conformational determinants. It is the thesis of this paper that a string of three amino acid residues (a tripeptide) is antigenic when it is located on a large carrier, that is, when it is part of a protein. Conceptually this has great explanatory power in understanding (a) autoimmune phenomena (b) the intriguing finding that monoclonal antibodies which are supposed to be exquisitely specific cross-react with disparate, non-homologous proteins. Clinical syndromes such as the neuropathies of myeloma, hepatitis and multiple sclerosis are discussed in the light of this concept by computer analysis of the putative antigenic sites of myelin basic protein, hepatitis B and A proteins and measles peptides.

The radioimmunoassay of human serum albumin with antibody adsorbed on porous glass beads.

A convenient method for the radioimmunoassay of human serum albumin (HSA) is presented. It utilises porous glass beads on which rabbit anti-HSA has been strongly adsorbed. Assay time is less than an hour: no centrifugation is required and the range is 50--1,000 ng ml-1.

The molecular biology of cancer and its diagnostic implications.

The origin of cancer is discussed from the view of the two-stage model of malignant transformation. Environmental carcinogens play an integral part in the process. When the cell is transformed, cell surface changes are found for such components as fibronectin, collagen, actin, myosin, glycopeptides and enzyme activities. Hormone receptors are a fruitful line for research. Both qualitative and quantitative alterations are also seen with cancer cell enzymes. Among enzymes that can be used as markers of malignancy are the protease. A group of oncodevelopmental proteins, hormonal and non-hormonal, are in regular service for the management of cancer. Improvements in diagnostic specificity can be expected as the newer technologies are harnessed for medical use.

Tissue polypeptide antigen (TPA) and beta 2-microglobulin (beta 2-MG) as tumour markers in nasopharyngeal cancer.

The levels of tissue polypeptide antigen (TPA) and beta 2 microglobulin (beta 2-MG) were measured in the serum of 38 patients with nasopharyngeal carcinoma (NPC). Using sera from 35 normal volunteers and blood donors, a normal range for these two tumour markers was established. The normal range for TPA was 40-148 IU/L (mean = 93), while that for beta 2-microglobulin was 0.9-2.0 mg/L (mean = 1.3). In the patients with NPC but without known metastases the range was 63-178 IU/L for TPA (mean = 111) and for beta 2-MG, the range was 1.0-3.1 mg/L (mean = 1.7). For those NPC patients with metastases to bone or liver, the mean TPA was 464 IU/L and the mean beta 2-MG was 4.3 mg/L. It appears that TPA and beta 2-MG are useful markers for the monitoring of NPC patients for metastatic disease, particularly TPA.

Tumour markers--a diagnostic and therapeutic perspective.

A number of chemically diverse substances is associated with the malignant transformation of cells. Though none of these can be said to be wholly specific they are operationally useful and this paper is a selective review of some of them. They arise from exposure of previously cryptic epitopes in membranes and secreted molecules and the amplification of normally small quantities of cellular components, such as oncogenes in chromosomes. Antibodies directed to these tumour markers are opening up new avenues of research, diagnosis and therapy of cancer patients. The use of radioisotope labelled antibodies has become sufficiently sensitive and specific to be useful for decision-making in their clinical management.

Use of radionuclides in clinical endocrinology.

Radionuclides provide sensitive reporter molecules for labeling pharmaceuticals. Hormone measurements using radioimmunoassays are commonplace today, increasing our understanding of the pathophysiology of endocrine disease. In turn, hormones tagged with radionuclides are opening studies on new fields of receptor defects both at the receptor site and beyond. Common disorders such as diabetes mellitus, hyperthyroidism and acromegaly have been the first to benefit. Tracer methods to study secretory and clearance rates and the size of metabolic pools are mainly based on the use of radionuclides. DNA probes are useful in unravelling endocrine defects at the genome level. Originally using radioiodine but now an increasing number of newly synthesised radiopharmaceuticals, the individual organs are being visualised more specifically. Among these agents are labelled monoclonal antibodies hunting for neoplasias and analogs of adrenal medullary hormones such as metaiodobenzylguanidine (MIBG). From these studies the therapy of endocrine disorders will ultimately benefit.

Postmortem endocrine levels in the vitreous humor.

The vitreous humor and sera from 51 autopsy cases were assayed for progesterone, estradiol, thyroxine, triiodothyronine, TSH, FSH, LH and prolactin. The results indicate that the blood retinal barrier has unique properties of its own. All hormones were measurable in the sera. Progesterone, estradiol, T3 and T4 were not detected despite their lipid solubility and their small molecular size while the larger glycoprotein hormones were easily measureable.

Estradiol binding capacity of breast cancer cell line in correlation with cell cycle.

The specific estrogen binding capacity of the MCF-7 cells has been studied throughout the cell cycle. Two days after plating 1-4 X 10(5) cells per well, we observed that cell number, protein concentration and DNA level began to rise but the estrogen binding capacity was lowered. At logarithmic phase the binding increased gradually then sharply to just before confluency. When the cells reached the quiescent phase the estrogen binding capacity fell rapidly. Since estrogen binding capacity of the intact MCF-7 cells varies with different phases in the cell cycle as well as the incubation temperature and time and the plating density, all these factors should be taken into consideration when this cell line is used for hormone studies.

Quality control of estrogen and progesterone receptor assays.

The intra-laboratory QC materials for estrogen and progesterone receptor assays were prepared using different methods. The most convenient and economical method was found to be the use of lyophilized rat uterine cytosol. The level of receptors remained stable for 12 months when stored at -70 degrees C with the inclusion of 10 mM phenylmethyl sulfonyl-fluoride in the cytosol extraction buffer. For inter-laboratory comparison, we received QC materials from Dr E D Ryan (McMaster University, Canada) monthly and our results fell within +/- 1SD of the mean value of 15 centres from North America and Europe participating in this program.