M. Dietel

Morphological detection and quantification of lipoprotein(a) deposition in atheromatous lesions of human aorta and coronary arteries

Lipoprotein(a), as an atherogenic particle, represents an independent risk factor for coronary heart disease. In the present study the morphological distribution of apoprotein (a) and apoprotein B within the arterial wall is described. Apoprotein B, a constituent of very low-density lipoprotein, low-density lipoprotein and lipoprotein(a) has previously been demonstrated in atheromatous lesions. Lipoprotein(a) possesses an additional protein, designated apoprotein (a). Autopsy material (n = 74) from the left coronary artery and from the thoracic aorta has been examined by means of immunohistochemistry and both apoprotein (a) and apoprotein B were detected, primarily associated with the extracellular matrix and accumulating in lesions in the arterial wall. The staining pattern for both antigens was almost always found to be congruent, suggesting that the detection of (a)-antigen has to be attributed at least in part to the presence of lipoprotein(a). It is concluded that both low-density lipoprotein and lipoprotein(a) have an important role in the pathogenesis of atherosclerosis.

Nuclear DNA content of borderline tumors of the ovary: correlation with histology and significance for prognosis

Scanning-DNA cytophotometry was applied to Feulgen stained sections of 22 borderline tumors of the ovary (BOT). The DNA content was related to conventional histology. In 11 cases clinical follow up for more than 5 years was available. The DNA measurements disclosed two subgroups in the group of BOT. One showed a nuclear DNA content not exceeding tetraploidy (4c) indicating proliferative activity without malignant change and a second one exhibited DNA values higher than 4c indicating malignant transformation. Correlation of histological evaluation with the DNA content revealed a good agreement in 15 cases. However, a discrepancy was found in 7 cases: either the histological evaluation aroused suspicion for malignant potential but the histogram showed DNA values not higher than 4c (n=4), or histology showed well differentiated lesions with an atypical histogram (n=3). Clinical monitoring revealed no recurrence or tumor spread in all but one case of the group of lesions with DNA values up to 4c, whereas in the group with atypical DNA histograms (DNA values>4c) relapse appeared in 6 out of 7 cases. The results suggest that DNA analysis has prognostic significance for BOT.

Effect of continuous vs intermittent application of 3-OH-tamoxifen or tamoxifen on the proliferation of the human breast cancer cell line MCF-7 M1

SummaryThe antiproliferative potency of 5x10-7M tamoxifen (TAM) and 3-hydroxytamoxifen (3-OH-TAM) was investigated during continuous (8 days) or intermittent (2 h every 2nd or 3rd day, respectively) application to the oestrogen-receptor-positive, estradiol-sensitive human mammary carcinoma cell line MCF-7 M1, a variant of MCF-7 wild type. Growth modulation was evaluated in parallel by counting cells and by measuring DNA content. Continuous incubation resulted in a growth inhibition to 21.8±3.2% by 3-OH-TAM and to 39.5±4.8% by TAM when compared with control cultures defined as 100%. Intermittent addition induced a growth reduction to 23.0±2.1% by 3-OH-TAM and to 41.2±2.4% by TAM in relation to 100% controls. Addition of 3-OH-TAM for 2 h only at day 1 resulted in an inhibition to 70.3±3.2%, again in relation to 100% controls. When TAM was administered once for 2 h at day 1 it induced an inhibition to 79.0±4.9% at day 8. The in vitro results indicate that (a) at 5x10-7M3-OH-TAM has a better antiproliferative effectiveness than TAM, (b) the intermittent application is as effective as continuous application (no significant difference), and (c) the addition once a week reveals only a slight growth reduction after 8 days of culture.Application of the long-living TAM results in continuously high serum concentrations, which have been shown to create resistant cell clones. Compared to TAM the 3-OH metabolite has a considerably shorter half-life and its application in vivo reveals rise and fall of its serum concentrations. Since the presented data demonstrate that 3-OH-TAM is more potent than TAM and that the intermittent application is as effective as the continuous form, interval therapy with 3-OH-TAM may slow down the process of acquiring resistance to antioestrogens.

Pancreatic endocrine carcinoma with ectopic PTH-production and paraneoplastic hypercalcaemia

In a case of pancreatic endocrine carcinoma hypercalcaemia without bone metastases and normal parathyroid glands prompted our suspicion that there was paraneoplastic production of an osteoclast activating substance by the tumour tissue. This view was further confirmed by bone histology. Immunohistology post mortem revealed the production of PTH in the primary tumour and a liver metastasis. The usefulness of immunohistology in detecting paraneoplastic secretion of hormonal substances is discussed.

Establishment of primary cell cultures: Experiences with 155 cell strains

SummaryCell culture systems allow the examination of cell populations in a functional state. To simulate in vivo conditions as closely as possible freshly established cell strains are superior to permanent cell lines. Different aspects for the establishment of primary cell cultures obtained from various tissues are compared: (1) Disintegration, (2) culture media supplemented with basal additions, (3) special supplements (growth factors, hormones), and (4) attachment factors. The proliferation rates of the attained cell strains were evaluated by determination of cell doubling times. Procedures for how to obtain a relatively high plating efficiency (approx. 70% in our series of 219 attempts) of primary growth in vitro are described: (1) Mechanical disintegration is superior to enzymatic digestion. If mechanical treatment alone did not produce a sufficient number of viable cells, additional digestion with collagenase/dispase revealed a higher number of proliferating primary cultures than with trypsin. (2) Proliferation of cell cultures from normal and tumorous tissues of epithelial origin was superior in Leibovitz L 15 medium (58 of 87 (67%) cases). Cultures from mesenchymal tissues and tumors were found to have shortest cell doubling times in MEM and RPMI 1640 (16 of 23 (70%) cases). The media were supplemented with the basal additions indicated. (3) In approx. 30% of the cases special supplements like growth factors or hormones increased cell replication, although they were almost always not essential for cell growth. (4) Attachment factors only rarely contributed to the initiation of primary monolayer cultures. The application of various culture conditions does not lead to a protocol optimal for all tissues, for all probes of the same type of tumor, or for all tumor specimens of unique differentiation.

Effectiveness of antineoplastic drugs on the proliferation of human mammary and ovarian carcinoma cells in monolayer culture.

SummaryProcedures for the in vitro determination of the drug-induced inhibition of mammary and ovarian carcinoma cell growth were established. In monolayer cultures derived from advanced tumors, separation of epithelial carcinoma cells from concomitant cells of fibroblast-like or mesothelial appearance was achieved by differential trypsinization. The carcinoma cell character of the stock cultures was verified by chromosome analyses showing a high degree of aneuploidy for the epitheloid cell lines and euploidy for cells of apparently mesenchymal origin. When cultured carcinoma cells were injected in nu/nu mice, the tissue and cell cultures obtained from the heterotransplantation tumors closely resembled the original tumors and cell cultures in morphology, karyotype, and expression of tumor markers. The action of carcinostatic drugs in the logarithmic phase of the carcinoma cell proliferation was tested by kinetics experiments in multiple experimental cultures. In cell proliferation assays based on cell counts the 50% inhibition dose (ID50) of the drug effects was determined from the dose-response curves. Comparison of the ID50s revealed highly differential effectiveness of the drugs examined. The inhibitory effects were reproducible, rendering the procedures used suitable for testing the chemosensitivity of newly explanted gynecological carcinoma cells by proliferation assays.

Alteration of steroid hormone sensitivity during the cultivation of human mammary carcinoma cells.

SummaryDuring early cultivation steps of the newly derived and karyotyped human mammary carcinoma line EFM-19, the cells developed faster growth rates and became increasingly less responsive to the presence of serum in the culture medium. No drastic alterations of the morphology and of the karyotype were observed, and carcinoembryogenic antigen remained expressed during the course of the cultivation. In experimental incubations at various time intervals after the explantation, the cell proliferation was analyzed for dose-dependent effects of estradiol, cortisol, progesterone, and testosterone. After 16 wk of cultivation of the stock culture in the presence of estradiol, the cells had acquired a distinct sensitivity to estradiol resulting in permanent growth enhancement. The withdrawal of cortisol from the medium of the stock culture subsequently provoked the loss of the initially noted stimulation of the proliferation by cortisol. The stimulatory effect of progesterone on the proliferation was reversed to inhibition when the stock culture was deprived of cortisol in the growth medium. The results indicate that the choice of steroid hormones in the stock culture medium was determining the quality of the cellular growth responses.

Correlation of electron microscopic and secretory response of human parathyroid adenomas with different calcium concentrations in organ culture.

Twelve parathyroid chief cell adenomas from patients with primary hyperparathyroidism were incubated in a tissue culture system in the presence of different calcium concentrations and for various time periods. The endocrine response of the tissue was examined electron microscopically and radioimmunologically. After incubation in a medium of low calcium concentration the parathyroid adenomas showed ultrastructural signs of stimulation with proliferation of the hormone-synthesizing organelles. The development of the ultrastructural response could first be observed after four hours and increased up to several days. Radioimmunologically, an increase of the hormone secretion could be demonstrated. Converse results were obtained after incubation of the tumor tissue under suppressive culture conditions. To check for de-novo synthesis of the hormone released the tissue was incubated in a 75Se-methionine-containing medium. This resulted in radioactivity of the secreted parathyroid hormone, indicating de novo synthesis in our culture system. The biological potency of the released hormone was demonstrated by comparison of the PTH out of the medium with the international human MRC standard using two different radioassays.

Effectiveness of mitoxantrone on the proliferation of cell cultures derived from malignant mesenchymal tumors of human origin.

SummaryThe antiproliferative potency of mitoxantrone (MITOX) has predominatly been established for epithelial and hematologic neoplasias. In this study the effectiveness of MITOX was investigated in vitro for 6 sarcomatous human cell lines derived from 2 synovial sarcomas, a malignant schwannoma, a malignant histiocytoma, a leiomyosarcoma, and a chondrosarcoma. The examination was performed using a proliferation assay with monolayer cell cultures. The effect of MITOX was compared with that of adriamycin (ADR) and cisplatin (CDDP). For each drug at least 3 concentrations were tested which covered the therapeutically achievable range, i.e., for MITOX 0.2–0.002 μg/ml, for ADR 0.5–0.005 μg/ml, and for CDDP 5.0–0.05 μg/ml. Test incubations were performed for 3 days. Antiproliferative potency of the cytostatic drugs was assessed by counting the number of cells at the start and the end of the test period with and without drug addition. Furthermore the dose inhibiting cell growth to 50% of controls (ID50) was determined for MITOX. For comparison 4 cell lines from carcinomatous lesions were included in the study. MITOX inhibited proliferation rates of 4 sarcomatous tumor cell lines more intensively than ADR, and was less effective in 2 cell lines. However, these differences were not significant. In all mesenchymal cell lines tested the antiproliferative potency of MITOX was more pronounced than that of CDDP. In carcinomatous cell lines the MITOX-induced growth inhibition was similar to that found in response to administration of ADR and CDDP confirming the described effect on epithelial tumors. The study suggests that MITOX possesses a growth inhibitory potency for malignant soft tissue tumors in vitro. From these data it may be worthwhile to initiate clinical trials testing the treatment of sarcomatous lesions with MITOX.