Subcloning of ovarian cancer cell lines.

Cellular heterogeneity of malignant tissues is a well-known phenomenon (1). Intralineal/intraclonal diversity may be explained in part by proposing the concept of a hierarchically ordered, differentiating and self-renewing stem cell system for transformed cell populations (2). However, in many solid tumors, the stem cells are not easily accessible to phenotypic identification. In the past, density gradient centrifugation was successfully used to separate cells from tumors and from cell lines into distinct subpopulations (3-5). Using Percoll density gradients, we isolated undifferentiated clonogenic tumor stem-cell fractions from HOC-7 human ovarian adenocarcinoma cells. In addition, we also identified a low-density cell subpopulation formed by large, vacuolated, slowly growing, adenoid differentiated cells with very low clonogenic activity (6-11). Further characterization of these cell fractions in terms of stability of the isolated phenotypes is essential for the assessment of their biological significance. Subcloning of the isolated cell fractions by limiting dilution culture (12) followed by long-term culture yielded three permanent monoclonal sublines, which reveal a stable adenoid differentiated phenotype, and three subclones representing undifferentiated, clonogenic tumor stem cells (13). These data demonstrate that the isolated phenotypes represent distinct cell entities reflecting specific stages of ovarian surface epithelial cell differentiation.

Activation of retinoic acid receptor alpha is sufficient for full induction of retinoid responses in SK-BR-3 and T47D human breast cancer cells.

Retinoid signaling via retinoic acid (RA) and retinoid X receptors (RARs and RXRs) regulates mammary epithelial cell growth and differentiation. Loss of RAR-beta might represent an early event during breast carcinogenesis. Higher differentiated, estrogen-dependent, estrogen receptor (ER)-positive (ER+) mammary carcinoma cells have been found to contain relatively high levels of RAR-alpha and to be responsive to retinoids, whereas most undifferentiated, estrogen-independent, ER-negative (ER-) cells are characterized by low RAR-alpha expression and by retinoid resistance. In contrast, RAR-gamma is detectable at equal levels in both ER+ and ER- cells. In the present investigation, we directly examined the relative contribution of the distinct retinoid receptors to the retinoid response of breast cancer cells by comparing the effects of low concentrations of specific retinoids, which selectively activate individual receptor subtypes, on growth, cell cycle distribution, apoptosis, and on the autoregulation of RAR-alpha and RAR-gamma in ER- SK-BR-3 and ER+ T47D breast cancer cells. In vitro growth activity was determined by using a colorimetric cell viability assay and analysis of cell cycle distribution, and apoptosis was performed by flow cytometry of propidium iodide-stained or fluorescent Annexin V-labeled cells, respectively, whereas expression of RAR-alpha and RAR-gamma was determined by Northern blotting. Both cell lines are retinoid sensitive and express high amounts of RAR-alpha, RAR-gamma, and RXR-alpha. RAR-alpha-selective compounds (AM80 and AM580) inhibit cell growth, induce G1 arrest, stimulate apoptosis, and up-regulate RAR-alpha and RAR-gamma mRNA as efficiently as RAR/RXR-pan-reactive (9-cis RA) and RAR-pan-reactive retinoids (all-trans RA, TTNPB). Remarkably, an RAR-alpha antagonist (Ro 41-5253) not only blocks the RAR-alpha-selective agonists but also the pan-reactive compounds. In contrast, RAR-13-selective (CD417), RAR-gamma-selective (CD437/AHPN), and RXR-alpha-selective (Ro 25-7386) retinoids exert no effects on the examined parameters. Thus, our results support the idea that RAR-alpha is the crucial receptor mediating the biological effects during retinoid signaling in both ER- SK-BR-3 and ER+ T47D human breast cancer cells.

Inhibition of proliferation and induction of apoptosis in soft tissue sarcoma cells by interferon-alpha and retinoids.

Uncontrolled proliferation and a defect of apoptosis constitute crucial elements in the development and progression of tumours. Among many other biological response modifiers known to influence these mechanisms, the efficacy of retinoids and interferons in the treatment of various malignant entities is currently matter of discussion. In the present study, we have investigated the effects of 9-cis-retinoic acid (9cRA), 13-cis-retinoic acid (13cRA), all-trans-retinoic acid (tRA) and interferon-alpha on proliferation and apoptosis of human soft tissue sarcoma (STS) cell lines HTB-82 (rhabdomyosarcoma), HTB-91 (fibrosarcoma), HTB-92 (liposarcoma), HTB-93 (synovial sarcoma) and HTB-94 (chondrosarcoma) in relation to p53 genotype as well as p53 expression. HTB-91, HTB-92 and HTB-94 STS cells exhibited mutant p53, whereas wild-type p53 was found in HTB-93 STS cells, and a normal p53 status in HTB-82 STS cells, carrying a silent point mutation only. Interferon-alpha, irrespective of p53 status, inhibited the proliferation of all five cell lines dose- and time-dependently. Similarly, 9cRA, 13cRA and tRA decreased the proliferation of HTB-82 and HTB-93 STS cells, whereas the proliferation of p53-mutated HTB-91, HTB-92 and HTB-94 STS cells remained unchanged. Furthermore, only 9cRA and tRA were capable of inducing apoptosis in HTB-82 and HTB-93 STS cells, whereas HTB-91, HTB-92 and HTB-94 STS cells did not undergo apoptosis under the influence of 9cRA or tRA. Retinoic acid receptor (RAR)-alpha and RAR-beta mRNA were not detectable by Northern blot analysis in the five STS cell lines, whereas mRNA for the universal retinoic acid receptor, RAR-gamma, was expressed in all STS cell lines indicating that retinoid resistance was not associated with a lack of RAR expression. Apoptosis was not induced by interferon-alpha or 13cRA in any of the five STS cell lines tested. Our results indicate that within the panel of tested STS cell lines, inhibition of proliferation and induction of apoptosis result from different mechanisms which differ in their dependence upon the presence of intact p53.

Expression of c-erbB-4/HER4 is regulated in T47D breast carcinoma cells by retinoids and vitamin D3.

Nuclear steroid/retinoid and memgbrane c-erbB receptor tyrosine kinase signaling control proliferation and differentiation of mammary epithelial cells. Recently, we reported that retinoic acids are efficient repressors of c-erbB-2 and -3, but not of c-erbB-1 gene expresson. Here we demonstrate that retinoid acid- mediated growth inhibition is accompanied with reduced expression of c-erbB-4/HER4 in T47D breast cancer cells as determined by FACS, Western, and RT-PCR. All-trans and 9-cis retinoic acid reduce c-erbB-4 expression to 30%-60% of control, depending on the concentration. Dexamethasone (Dex) is inactive on D3 (D3), in contrast, acts as a strong inducer, elevation more that twofold at the mRNA, but does not significantly affect cell growth. We concolude that retinoic acids are efficient growth inhibitors and repressors of cell growth and c-erbB-4, whereas D3 represents a highly efficient inducer of c-erbB-4 expression with affecting cell proliferation.

Involvement of nuclear steroid/thyroid/retinoid receptors and of protein kinases in the regulation of growth and of c-erbB and retinoic acid receptor expression in MCF-7 breast cancer cells.

Nuclear steroid/thyroid/retinoid receptors and c-erbB membrane receptor tyrosine kinases control epithelial growth and differentiation. Retinoid receptors can dimerize with the vitamin D receptor, the glucocorticoid receptor or the thyroid receptor. Furthermore, multiple c-erbB receptor dimers have been identified. It has been shown that some of these receptor pathways communicate with each other via cross-connected regulatory networks. Molecular interactions between retinoid receptors or estrogen receptors (ER) and c-erbB-2, and between ER and retinoic acid receptor(RAR)-alpha have been reported. Here, we demonstrate the effects of steroids/thyroids/retinoids and of activators of protein kinase A (forskolin, Forsk) and C (12-O-tetradecanoylphorbol-13-acetate, TPA), on growth and expression of c-erbB and RARs in MCF-7 breast cancer cells, which contain high levels of RAR-alpha and -gamma, and which express significant amounts of c-erbB-2 and -3. All trans-retinoic acid (tRA), the anti-estrogen ICI 182 780 (ICI), Forsk and TPA reduced, whereas triiodothyronine and 17beta-estradiol (E2) stimulated cell growth. Flow cytometry revealed that tRA and E2 reduced c-erbB-2 and -3, whereas tamoxifen, Forsk and TPA up-regulated c-erbB-2. c-erbB-3 was co-regulated with c-erbB-2. Northern analysis demonstrated that RAR-alpha was down-regulated by dexamethasone, ICI, and TPA, whereas vitamin D3 and E2 up-regulated RAR-alpha. RAR-gamma expression was less responsive to such treatment, being reduced only by ICI and Forsk. These data indicate that nuclear receptor and protein kinase signaling communicate with each other and control the expression of RARs and c-erbB receptors. Efficient growth control requires the coordinated interplay of both receptor systems.

Retinoids control the expression of c-erbB receptors in breast cancer cells.

Nuclear retinoid and membrane c-erbB receptors participate in signal transduction systems that control mammary epithelial cell proliferation and differentiation. Recently, we demonstrated that c-erbB receptor activation stimulates retinoic acid receptor-alpha expression. We now report that retinoids reduce SK-BR-3 breast cancer cell growth by inhibiting the cell cycle and by inducing apoptosis. This is accompanied with reduced c-erbB expression as determined by FACS, Western, Northern, RT-PCR, and reporter assays. All-trans (ATRA) and 9-cis retinoic acid (9cRA) reduce c-erbB-1 protein to 50-100%, c-erbB-2 to 20-30%, and c-erbB-3 to 10-50% of control, depending on the concentration, respectively, without influencing the tyrosine phosphorylation status. Down-regulation of c-erbB-2 and -3 was seen at all levels analyzed, whereas c-erbB-1 mRNA remained unchanged. Retinoic acid-mediated down-regulation of growth and c-erbB-2 and -3 expression was also seen in MCF-7 cells. We conclude that retinoic acids are efficient repressors of c-erbB-2 and -3 gene expression, whereas c-erbB-1 is not markedly affected.

Tyrosine kinase signaling pathways control the expression of retinoic acid receptor-alpha in SK-BR-3 breast cancer cells.

Breast carcinomas are frequently characterized by hyperactivated c-erbB receptor tyrosine kinase signaling. Combination of anti-proliferative retinoids with growth-inhibitory c-erbB-specific agents might induce therapeutic benefit. We demonstrate close interactions between the c-erbB and the retinoic acid receptor system in SK-BR-3 breast cancer cells. Epidermal growth factor and heregulin-beta1 activate c-erbB receptors and dose- and time-dependently up-regulate retinoic acid receptor-alpha (RAR-alpha) mRNA. Similar effects have been found for the growth-inhibitory c-erbB-2 receptor tyrosine kinase-activating antibody 4D5 and the tyrosine phosphatase inhibitor orthovanadate. In contrast, the tyrosine kinase-inhibitor herbimycin A reduces tyrosine-specific protein phosphorylation and down-regulates RAR-alpha. Our data demonstrate that the expression of RAR-alpha, which represents a key mediator of the anti-proliferative effects of retinoids in breast cancer cells, is regulated by modulators of tyrosine kinase signaling. The levels of RAR-beta and -gamma mRNAs, however, are not affected by such agents.

Regulation of estrogen receptor concentration and activity by an erbB/HER ligand in breast carcinoma cell lines.

Expression of the erbB-2 oncogene in breast cancer patients correlates with poor prognosis and failure of hormonal therapy. In this study, the effects of a putative erbB/HER ligand, gp30, on estrogen receptor (ER) concentration and activity was investigated in the estrogen receptor positive human breast cancer cells, BT474 and MCF-7, which express either high or low levels of erbB-2 and erbB-4, respectively. Treatment of cells with gp30 resulted in a decrease in the steady-state level of estrogen receptor protein by approximately 70-80%. The effect of gp30 on the concentration of ER was independent of serum in the media and was not inhibited by an epidermal growth factor receptor blocking antibody. In addition to the effect on ER protein, gp30 decreased the steady-state level of ER messenger RNA. Transcription run on experiments demonstrated that the decrease in ER expression was mediated by a decrease in ER gene transcription. The effect of gp30 on estrogen receptor activity was also investigated in this study. Treatment of cells with gp30 blocked estradiol induction of progesterone receptor. Inhibition was observed at the level of progesterone receptor protein, messenger RNA, and gene transcription. gp30 also blocked estradiol induction of pS2 gene transcription. In addition to its effects on progesterone receptor and pS2, gp30 blocked activation of an estrogen response element in a transient transfection assay and inhibited ER binding to its response element in a DNA mobility shift assay, suggesting a direct effect on the estrogen receptor. The effects of gp30 on estrogen receptor concentration and activity were independent of the level of erbB-2 and erbB-4 in the cell. These data show that gp30 regulates the concentration of ER and modulates ER activity.

Bidirectional interactions between the estrogen receptor and the cerbB-2 signaling pathways: heregulin inhibits estrogenic effects in breast cancer cells.

The responsiveness of estrogen receptor (ER)-positive breast cancer to endocrine therapy is frequently reduced in cells over-expressing c-erbB-2. Stimulation of ER suppresses c-erbB-2, indicating that estrogen controls the activity of c-erbB-2. Heregulin (HRG) has been described to bind to c-erbB-3/c-erbB-4 and to stimulate c-erbB-2. Here we describe the effects of HRG on cell growth and on ER and c-erbB-2 expression in breast cancer cell lines containing distinct levels of c-erbB-2 and ER (BT-474: c-erbB-2 , ER+; MDA-MB-361: c-erbB-2++, ER++; MCF-7: c-erbB-2+, ER ). Proliferation of estrogen-stimulated, c-erbB-2 and ER-positive cells is inhibited by HRG in a dose-dependent manner. In addition, HRG dose-dependently inhibits ER expression. Estrogen, however, inhibits c-erbB-2. Estrogen-mediated down-regulation of c-erbB-2 is most pronounced in MCF-7 but weaker in BT-474. In the latter cells HRG efficiently blocks the estrogenic effect on c-erbB-2. In MCF-7 cells, however, the inhibition of c-erbB-2 cannot be completely reverted by HRG. This modulation occurs in all 3 cell lines at protein, RNA and transcriptional levels, suggesting that the activity of the c-erbB-2 promoter, which contains an estrogen-responsive region, is affected by HRG. The intensity of the mutual inhibition between the HRG/c-erbB-2 and the estrogen/ER system depends on the relative levels of ER and c-erbB-2 expression in the respective cell lines.

Retinoic acid receptors in retinoid responsive ovarian cancer cell lines detected by polymerase chain reaction following reverse transcription.

The growth inhibitory effects of all-trans and 13-cis retinoic acid (RA) and of the synthetic retinoids TTNPB, TTNPB-ethylester and TTNN were studied on seven human epithelial ovarian cancer cell lines and one ovarian teratocarcinoma cell line. Six of seven ovarian adenocarcinoma cell lines were inhibited in their growth by RA and by synthetic retinoids in a dose dependent manner. No response to these substances was observed for the ovarian teratocarcinoma cell line. The knowledge that RA and retinoids exert their action on the cells via nuclear receptors led us to examine the expression of RAR-alpha, -beta and -gamma mRNA by these cell lines by polymerase chain reaction following reverse transcription. All cell lines expressed RAR-alpha and -gamma mRNA and six of the eight cell lines were found to express additionally RAR-beta mRNA, among them the ovarian teratocarcinoma cell line. Our data indicate that there was no direct association between the presence of RAR subtype transcripts and the response to retinoids in ovarian cancer cell lines.

Modulation of the immunophenotype of ovarian cancer cells by N,N-dimethylformamide and transforming growth factor-beta 1.

Exposure of HOC-7 ovarian adenocarcinoma cells to regulators of cell differentiation caused inducer-dependent alterations of the antigenic pattern of the cells. Immunocytochemistry revealed that N,N-dimethylformamide (DMF) elevated the membrane staining for epidermal growth factor (EGF)-receptor and for desmoplakins I and II. DMF also stimulated cytoplasmic and surface labeling for CA 125 and the deposition of fibronectin into the extracellular matrix. Stimulation of fibronectin was also seen after addition of transforming growth factor (TGF)-beta 1. These responses were quantified using a fixed-cell, enzyme-linked immunosorbent assay (ELISA) and revealed that DMF dose-dependently induced expression of EGF-receptor, CA 125, fibronectin, and desmoplakins I and II. TGF-beta 1 stimulated fibronectin and desmoplakins I and II only. Production of EGF and TGF-alpha was not affected by these inducers. Immunocytochemistry, ELISA and Western blotting showed that both inducers caused down-regulation of myc oncoproteins. DMF was more effective in changing the immunophenotype of HOC-7 cells than TGF-beta 1. Desmoplakins I and II demonstrated elevated epithelial differentiation, whereas fibronectin indicated stimulation of extracellular matrix formation. Elevated EGF-receptor could not compensate for the growth inhibition induced by DMF. The expression of myc oncoproteins was inversely related to cell proliferation. CA 125, however, seems to be unrelated to cell growth.

Different propensity for spontaneous differentiation of cell clones isolated from the human ovarian surface epithelial cell line HOC-7.

Limiting dilution culture of cell fractions obtained by discontinuous density gradient centrifugation was used to establish six different cell clones from HOC-7 ovarian adenocarcinoma cells (D1-D3, N1-N3). Clones D1-D3 revealed a phenotype similar to that seen in parental cells exposed to differentiation inducers such as dimethyl sulfoxide (DMSO, 0.8% [v/v]). They were flattened, slowly growing cells (doubling times: 42-46 h). The cells developed long cytoplasmic extensions and adopted a complicated growth pattern. Fixed-cell enzyme-linked immunosorbent assay (ELISA) and Western blotting demonstrated that these cells contained high levels of epidermal growth factor-receptor (EGF-R), carbohydrate antigen 125 (CA 125), fibronectin and desmoplakin, but low levels of myc oncoproteins. However, untreated parental cells and clones N1-N3 were fast-growing (doubling times: 23-28 h), regularly shaped, polygonal cells ("cobblestone" monolayer) with low levels of EGF-R, CA 125, fibronectin and desmoplakin, but relatively higher amounts of myc oncoproteins. The similarity of the sublines to either untreated or inducer-treated parental cells indicated that clones D1-D3 represented spontaneously differentiated HOC-7 cells, whereas clones N1-N3 originated from less-differentiated cells. The features examined in this model cell system proved to be closely related to ovarian cancer cell proliferation and differentiation. The observation of a tumor-inherent propensity for spontaneous differentiation suggests that exogenous stimulation of existing differentiation pathways may represent an alternative approach for tackling the problem of growth control and differentiation in malignant tissues.

Comparative analysis of the effects of dimethyl sulfoxide and retinoic acid on the antigenic pattern of human ovarian adenocarcinoma cells.

HOC-7 malignant ovarian surface epithelial cells have been exposed to differentiation promoters like dimethyl sulfoxide (DMSO) and retinoic acid (RA) and the resulting cell phenotypes were characterized immunologically. Immunocytochemistry revealed that DMSO caused elevation of membrane-associated staining for epidermal growth factor-receptor (EGF-R) and for desmoplakins I and II (DPI+II). DMSO also stimulated cytoplasmic and surface labelling for CA 125 and extracellular deposition of fibronectin (FN). A fixed-cell ELISA system was used for quantification of these differentiation-like responses and revealed that DMSO efficiently induced expression of EGF-R, CA 125, FN and DPI+II in dose-dependent manner. Immunocytochemistry, ELISA and Western blotting additionally demonstrated that both DMSO and RA caused down-regulation of myc oncoproteins. Densitometer evaluation of electrophoresed proteins revealed a 50% DMSO- and a 25% RA-induced myc reduction. Apart from growth reduction, which was seen for both inducers, inhibition of myc gene expression was the only response of HOC-7 cells to RA-treatment. The extent of myc down-regulation seems, therefore, to be crucial for the initiation of maturational processes in the cells. Subsequent phenotypic differentiation of HOC-7 cells causes elevated levels of EGF-R, CA 125, FN and DPI+II. This cell model might be useful for the distinction between induced growth reduction and differentiation of ovarian cancer cells.

Relationship of myc protein expression to the phenotype and to the growth potential of HOC-7 ovarian cancer cells.

In this investigation we demonstrate expression of myc oncoproteins in HOC-7 ovarian adenocarcinoma cells. The cells were exposed to differentiation inducing agents such as dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), retinoic acid (RA) and transforming growth factor-beta 1 (TGF-beta 1). Myc protein expression in treated cells was then compared with that in control cultures and in monoclonal HOC-7 sublines, which are characterised by distinct phenotypes. Cells exposed to DMSO and DMF became markedly enlarged and flattened and developed cytoplasmic extensions. They looked similar to a subline, which revealed a less malignant and more differentiated cell phenotype. All four inducers prolonged the cell doubling time and reduced the saturation density to levels, normally found in the more differentiated subline. Furthermore, all inducers except RA elevated extracellular fibronectin, which is characteristic for less malignant epithelial cell phenotypes. All four agents inhibited myc oncoprotein expression reversibly (1% DMSO greater than 0.5% DMF greater than 10 microM RA greater than 10 ng ml-1 TGF-beta 1) and in time-dependent manner. Down-regulation of myc protein expression is, therefore, closely related to inducer-dependent growth reduction of HOC-7 cells and to the development of a less malignant cell phenotype.

The differential effects of N,N-dimethylformamide and transforming growth factor-beta 1 on a human ovarian cancer cell line (HOC-7).

We have compared the effects of N,N-dimethylformamide (DMF) and transforming growth factor (TGF)-beta 1 on the growth and phenotype of HOC-7 ovarian cancer cells. Previous density gradient fractionation of untreated HOC-7 cells suggested that rapidly growing small polygonal medium density cells revert spontaneously into less malignant flattened low density cells. Here we demonstrate that DMF and TGF-beta 1 induce similar flattened cell phenotypes. Both agents induce qualitatively similar alterations in the cells. DMF, however, exerted stronger effects than TGF-beta 1. The cells become flattened, develop cytoplasmic extensions, and reduce DNA-synthesis as well as anchorage-dependent and -independent growth. These effects are reversible after removal of the inducers, indicating that the cells have not become terminally differentiated. Electron microscopy demonstrates prominent filament bundles in treated cells. Immunofluorescence further shows that these cells contain large amounts of cytokeratin. Immunocytochemistry and ELISA demonstrate 1- to 5-fold higher amounts of desmoplakin and fibronectin after DMF- or TGF-beta 1-exposure. The described differentiation-like responses of HOC-7 cells can be used for recognition of pharmacologically induced maturation of ovarian cancer cells.

The effects of dimethyl sulfoxide and retinoic acid on the cell growth and the phenotype of ovarian cancer cells.

We have compared the in vitro effects of the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA) on a polyclonal human ovarian cancer cell line (HOC-7). Density gradient fractionation of untreated cells reveals that a proportion of rapidly growing, polygonal cells with medium density is capable of spontaneous reversion into a slowly growing low-density phenotype with flattened morphology similar to non-transformed human ovarian surface epithelial cells. Clonal expansion of these low-density cells proves that the observed characteristics are stable for prolonged culture periods. Exposure of HOC-7 cells to DMSO and RA or removal of the serum from the medium is effective in enhancing the proportion of these low-density cells. Application of DMSO causes the cells to become flattened and elongated, and to develop rod-like protrusions. In these cytoplasmic extensions thick filament bundles are dominant. Immunofluorescence studies demonstrate that both untreated low-density subclones and DMSO-treated polyclonal cells are much more reactive for cytokeratin than medium-density subclones or untreated parental cells. Furthermore, immunocytochemistry and fixed-cell ELISA reveal 2- to 5-fold greater amounts of desmoplakins I and II and of fibronectin in low-density subclones and in DMSO-treated cells as compared to medium-density subclones and control cultures. RA exerts weaker effects on the phenotype of the cells. Both inducers reduce DNA synthesis and inhibit the anchorage-dependent and the anchorage-independent cell growth in a dose- and time-dependent manner. The restoration of the original morphology and growth rate after removal of the differentiation-inducing agents proves that the observed changes are reversible; this indicates that the cells do not become terminally differentiated.(ABSTRACT TRUNCATED AT 250 WORDS)

Separation of clonogenic and differentiated cell phenotypes of ovarian cancer cells (HOC-7) by discontinuous density gradient centrifugation.

We isolated clonogenic cells from differentiated HOC-7 ovarian cancer cells. Both cell subsets were characterised in respect to morphology, growth behaviour, DNA content and expression of tumour-associated antigens and nuclear oncogenes. Ten cell fractions (Fr) were separated by centrifugation in a discontinuous density gradient (Fr 1 less than 1.037 g/ml to Fr 10 greater than 1.069 g/ml, steps 0.004 g/ml). Large adenoid cells containing vacuoles filled with neutral polysaccharides were concentrated in Fr 1-4. These cells were non-clonogenic in soft agar. The growth on solid substrate was highest in Fr 6 and 7, intermediate in Fr 2-5 and Fr 8-10 and lowest in Fr 1. The mean cloning efficiencies of the fractions in soft agar were highest in Fr 6 (8.1%) and lowest in Fr 2 and 3 (0.1%). Diploid and near tetraploid cell subsets were found with similar frequency in all fractions. Immunocytochemistry revealed 4-7% Ki-67 positive cells in Fr 1-6 and 12-20% in Fr 7-10. In Fr 3-10 greater than or equal to 79% of the cells expressed CA 125. Positivity for c-myc, c-myb and c-fos (greater than or equal to 74%) was not correlated with clonogenicity. In conclusion, differentiated cells (Fr 1-4) were separated from cells with higher growth rates (Fr 5-10). Clonogenic cells were enriched in Fr 6. These data indicate that discontinuous density gradient fractionation represents a useful method for separation of cells with different degrees of differentiation, growth potential and clonogenicity.

The angioarchitecture of the Lewis lung carcinoma in laboratory mice (a light microscopic and scanning electron microscopic study).

53 Lewis lung carcinomas implanted subcutaneously into C57BL/6-mice were examined. The animals were killed at various stages of tumor growth (TG) and prepared for histology and for scanning electron microscopy (critical-point-dried tissue; vascular corrosion casts). Prior to casting animals were rinsed using different perfusion pressures. Casting was done by manual injection of the resin, whereby different influx-rates were applied resulting in low, medium and high pressure preparations. We discern 3 phases of tumor angiogenesis (TA) occurring during 4 stages of TG among which vasodilation establishes the first reaction of the host vascular system to a growing tumor implant. During this stage 1 of TG, tumor nidation, nearby sinusoidal dilated host capillaries form globular outgrowings (phase 1 of TA). Subsequently radially arranged sprouts, which preferentially arise from venous host vessels, grow into the centre of the implant (phase 2 of TA). Stage 2 of TG, early tumor growth, is characterized by necrosis of the central tumor tissue and the development of a central avascular cavity. Thus the tumor vascular system is organized like a hollow sphere with a central cavity and a peripheral vascular "envelope" with large vessels embracing the tumor and centrifugally growing vascular sprouts, which arise from the venous part of the vascular "envelope" and invade the surrounding host tissue (phase 3 of TA). During stage 3 of TG, late tumor growth, many vessels of the basket-like vascular "envelope" obliterate. In stage 4 of TG, prefinal phase, the peripheral vascular density decreases continuously. Thus vascular sprouting and proliferation of viable tumor cells is confined to basal regions of the tumor.

The characteristic structural features of the blood vessels of the Lewis lung carcinoma (a light microscopic and scanning electron microscopic study).

Vascular corrosion casts of Lewis lung carcinomas (LLC) grown subcutaneously in C57BL/6-mice are correlated with histological sections and with tumor tissue prepared for scanning electron microscopy (SEM). By making low, medium and high pressure cast preparations we studied the influence of perfusion and injection pressure on the resulting cast sample. Three types of vascular proliferations are distinguishable in LLC: Small globular outgrowths on sinusoidal dilated tumor capillaries, caused by proliferation of their endothelial cells. New sprouts on surrounding host vessels, invading the small, still avascular implant. Superficially located, centrifugally running sprouts in peripheral regions of large tumors. They invade the surrounding host tissue. Vascular sprouts are of venous origin, have a fragmentary endothelium and are rather "leaky" if casted. High pressure preparations of large tumors reveal central avascular cavities surrounded by centripetally running, compressed and blind ending tumor vessels. Irrespective of the applied injection pressure, the casts always exhibit extravasal channels caused by degeneration of the endothelium of central tumor vessels. We show that SEM of vascular corrosion casts combined with histology not only demonstrates such contrary processes as the development of tumor blood vessels and the simultaneously occurring vascular degeneration, but also elucidates all other morphological characteristics of the tumor vascular system.

The vascular pattern of basal cell tumors: light microscopy and scanning electron microscopic study on vascular corrosion casts.

Light microscopy of hematoxylin-eosin stained tissue sections and scanning electron microscopy (SEM) of vascular corrosion casts were used to study the blood vascular system of human basal cell tumors. Concerning the gross angioarchitecture there is a very close correlation between the histological appearance and the findings obtained from vascular corrosion casts, when evaluated in a SEM. The tumor cell beds are enveloped by basket-like capillary plexus. The tumors are traversed over long distances by superficially running, teleangiectatic, but flattened capillaries. These compressed vessels are squeezed between tumor cell cords and epidermis. In vascular corrosion casts of human basal cell tumors the vascular system exhibits three different features. Blind-ending vascular casts; Four different causes for blind-ending cast structures are pointed out and discussed. Incomplete filling of the vascular system; compression of tumor vessels; new proliferating capillary sprouts; broken cast endings. Variations in vessel caliber and extravasation of the injection resin. Most of the variation in vessel calibers are thought to be caused by dilation of the weakened endothelial walls, but some of them presumably represent new projecting vascular swellings. Circumscribed leakage of the injected resin could be attributed to regions of advanced connective tissue degeneration and endothelial lysis. Flattened cast structures; The addition of tissues during tumor growth results in an increase of tissue pressure. Thus many tumor vessels get displaced, compressed, and flattened and vascular occlusions will occur. However, it must be stressed that much caution is needed in assessing the nature of the vascular cast structures of basal cell tumors.