Progestogens and membrane-initiated effects on the proliferation of human breast cancer cells.

OBJECTIVES: Evidence is accumulating that progestogens may play a crucial role in the development of breast cancer under contraception and hormone therapy in reproductive and menopausal women. Progesterone receptor membrane component 1 (PGRMC1) expressed in breast cancer may be important in tumorigenesis and thus may increase breast cancer risk. The aim of this project was to investigate the influence of progesterone and nine synthetic progestins on MCF-7 breast cancer cells overexpressing PGRMC1. METHODS: MCF-7 cells were stably transfected with PGRMC1 expression plasmid (WT-12). To test the effects of progestogerone (P) and the synthetic progestins chlormadinone acetate (CMA), desogestrel (DSG), drospirenone (DRSP), dydrogesterone (DYD), levonorgestrel (LNG), medroxyprogesterone acetate (MPA), nomegestrol (NOM) and norethisterone (NET) on cell proliferation, MCF-7 and WT-12 cells were stimulated with different concentrations (0.01-1 µmol/l). RESULTS: In MCF-7 cells, DRSP, DSG, DYD, LNG and NET increased the proliferation at 1 µmol/l, the effect being highest for NET with about 20%. In WT-12 cells, the same progestins, but additionally MPA, showed a significant increase, which was much higher (30-245%) than in MCF-7 cells. Here again, NET showed the highest proliferative effect. No effect was found for CMA, NOM and P. CONCLUSION: Some synthetic progestins trigger a proliferative response of PGRMC1-overexpressed MCF-7 cancer cells. The effect of progestogens on breast cancer tumorigenesis may clearly depend on the specific pharmacology of the various synthetic progestins.

Morphometry and ultrastructure of prismatic cristae in mitochondria of a crayfish muscle. A hypothesis of the structural principle.

Approximately 40% of the mitochondria in the sphincter muscle of the crayfish vas deferens have prismatic-type cristae. In cross section, the angular cristae have either triangular or rhomboid profiles which are surrounded by a hexagonal array of electron-dense dots. In longitudinal section, these mitochondria exhibit both thick and thin parallel lines, which represent cristae and filaments, respectively. It is postulated that the matrix of the prismatic-type mitochondria is packed with rodlets composed of an electron-dense core and a less dense shell. Close packing of these rodlets results in the regular hexagonal dot array. Deletion of fascicles of 3 or 4 rodlets results in spaces with triangular or rectangular cross sections. Lining of these spaces with membranes results in cristae with triangular or rhomboid cross sections.

Membrane-initiated effects of progesterone on proliferation and activation of VEGF in breast cancer cells.

OBJECTIVE: Progesterone influences mammary gland development and probably breast cancer tumorigenesis and functions by regulating a broad spectrum of physiological processes. We investigated receptor membrane-initiated actions of progesterone in MCF-7 breast cancer cells via progesterone receptor membrane component 1 (PGRMC1). DESIGN AND METHOD: The expression of PGRMC1 in breast cancer was verified by immune fluorescent analysis of paraffin sections. MCF-7 cells were transfected with PGRMC1 (wild type) or PGRMC1 variants. These cells were stimulated with a membrane-impermeable progesterone (P4) conjugate (P4-BSA-fluorescein isothiocyanate, P4-BSA-FITC, 10(-6) mol/l) or unconjugated progesterone (P4, 10(-6) mol/l) in the presence or absence of the progesterone receptor blocker RU-486 (10(-6) mol/l). Additionally, the effects on the expression of vascular endothelial growth factor A (VEGF-A) were determined using quantitative real-time polymerase chain reaction. RESULTS: PGRMC1 is perinuclearly localized in breast cancer cells. Western Blot analysis suggests that PGRMC1 is phosphorylated at serine 180. MCF-7-PGRMC1 (S180A) cells show an approximately 35% increase in proliferation after incubation with P4-BSA-FITC compared to MCF-7 control and MCF-7-PGRMC1 (wild type) cells. This effect cannot be blocked by RU-486. P4 reduced proliferation of MCF-7-PGRMC1 cells by approximately 10% compared to untreated controls. P4-BSA-FITC treatment led to a roughly three-fold activation of VEGF-A gene expression compared to MCF-7 cells. CONCLUSION: PGRMC1 is expressed in breast cancer tissue and mediates an RU-486-independent proliferative signal. It might also contribute to VEGF-induced neovascularization in tumor tissue. Thus, screening for PGRMC1 expression might be of interest to identify women with a higher expression of PGRMC1 and who might thus be susceptible for breast cancer development under hormone replacement therapy.

Signalling pathways: jack of all cascades.

The transcription factors that bind the c-fos promoter element SRE are targeted by multiple, independent signalling cascades; the identities of these signalling pathways and their modes of activation are being elucidated.

Protein synthesis inhibitors reveal differential regulation of mitogen-activated protein kinase and stress-activated protein kinase pathways that converge on Elk-1.

Inhibitors of protein synthesis, such as anisomycin and cycloheximide, lead to superinduction of immediate-early genes. We demonstrate that these two drugs activate intracellular signaling pathways involving both the mitogen-activated protein kinase (MAPK) and stress-activated protein kinase (SAPK) cascades. The activation of either pathway correlates with phosphorylation of the c-fos regulatory transcription factor Elk-1. In HeLa cells, anisomycin stabilizes c-fos mRNA when protein synthesis is inhibited to only 50%. Under these conditions, anisomycin, in contrast to cycloheximide, rapidly induces kinase activation and efficient Elk-1 phosphorylation. However, full inhibition of translation by either drug leads to prolonged activation of SAPK activity, while MAPK induction is transient. This correlates with prolonged Elk-1 phosphorylation and c-fos transcription. Elk-1 induction and c-fos activation are also observed in KB cells, in which anisomycin strongly induces SAPKs but not MAPKs. Purified p54 SAPK alpha efficiently phosphorylates the Elk-1 C-terminal domain in vitro and comigrates with anisomycin-activated kinases in in-gel kinase assays. Thus, Elk-1 provides a potential convergence point for the MAPK and SAPK signaling pathways. The activation of signal cascades and control of transcription factor function therefore represent prominent processes in immediate-early gene superinduction.

CD95 (APO-1/Fas) induces activation of SAP kinases downstream of ICE-like proteases.

Triggering of CD95 (APO-1/Fas) on different T- and B-cell lines resulted in the induction of a number of kinases (35 kDa, 38 kDa, 46 kDa and 54 kDa) that phosphorylate c-Jun and to a lesser extent Histone H1. Activation of these kinases was independent of protein biosynthesis and preceded apoptotic DNA degradation. The kinase activation pattern was specific for CD95 triggering since a variety of physical or chemical inducers of T- and B-cell apoptosis activated different kinases. The kinase activities at 46 and 54 kDa contained members of the stress-activated family of protein kinases (JNK/SAPK). Activation of the CD95-specific set of kinases was prevented by treating cells with the ICE-inhibiting peptide N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) or by overexpression of the cow pox virus serpin CrmA. However, despite inhibition of ICE-like proteases the death signal was readily initiated at the cell membrane since a CD95 death-inducing signaling complex (DISC) was formed. Thus, our results demonstrate that ICE-like proteases in the CD95 pathway function downstream of the DISC but upstream of SAP kinases.

A phase I clinical trial of recombinant interleukin-2 by periodic 24-hour intravenous infusions.

Recombinant interleukin-2 (rIL-2) (NSC# 600664; Hoffmann-La Roche, Inc., Nutley, NJ) was studied in a phase I clinical trial in 33 patients with advanced, measureable cancer of the colon or malignant melanoma, Eastern Cooperative Oncology Group (ECOG) performance status O-1, and no prior chemotherapy or radiotherapy. The goal of the study was to identify a dose and schedule of IL-2 to generate maximal immune modulation with tolerable toxicity. Such a regimen might allow the addition of other treatment modalities and/or prolonged treatment duration in later trials. Each patient received IL-2 as a continuous 24-hour infusion once weekly for 4 weeks and then twice weekly for 4 weeks. Five treatment groups received from 10(3) U/m2 to 3 x 10(7) U/m2 per 24-hour infusion. The maximal tolerated dose was 3 x 10(7) U/m2/d twice weekly. Patients treated twice weekly at 1 x 10(7) and 3 x 10(7) U/m2/d had immune modulation in terms of lymphocytosis, eosinophilia, increased natural killer (NK) activity, and elevated numbers of peripheral blood mononuclear cells expressing CD16, OKT10/Leu-17, and Leu-19 surface markers. Endogenous generation of peripheral blood lymphokine-activated killer (LAK) activity was demonstrated by lysis of NK-resistant Daudi targets, in patients treated at 3 x 10(7) U/m2/d. Biochemical and hematological abnormalities were moderate and reversible. Clinical toxicity included hypotension, myalgia, arthralgia, stomatitis, fever, fatigue, nausea, headache, chills, diarrhea, and oliguria at high doses. Cardiovascular toxicity was tolerable for most patients and reversed after IL-2 was stopped. Two of six melanoma patients at 3 x 10(7) U/m2/d achieved partial responses by the end of the eighth week. This IL-2 schedule appears to produce potentially clinically useful immune enhancement with tolerable toxicity.

Differential and quantitative molecular analysis of ischemia complexity reduction by isotopic labeling of proteins using a neural embryonic stem cell model.

The analysis of rapid changes of protein expression in living systems in response to insults requires rigorous methods of complexity reduction. To control dynamic pattern of hundreds or even thousands of protein isoforms, we applied a novel method of differential molecular analysis to a cellular model which is suited to study ischemia. Neural derivatives of murine embryonic stem cells were exposed to chemical ischemia. The model was used to obtain starting material for a quantitative differential proteomics analysis. Fractionation of phosphoproteins from these samples and subsequent identification by mass spectrometry of differential proteins provide proof of principle of how novel molecular analytical tools provide new insight into the network of neuroprotective molecular events during specific situations of neuronal stress and related pharmaceutical intervention. Our results indicate a particular role of an isoform of the acidic calcium-independent phospholipase A2 in this type of insult.

c-Fos transrepression revisited.

The c-fos proto-oncogene was discovered by homology to transforming viral genes, leading to speculation that transforming viruses had captured a cellular gene involved in cell cycle control. Indeed overexpression of c-Fos protein led to deregulated growth control, and c-Fos was thought to be so critically involved in cell cycle control that transcriptional transrepression of its own promoter was interpreted as a negative feedback mechanism. However, recent findings render this conclusion improbable, Fos transrepression being most parsimoniously explained as transcriptional squelching imposed by artificially elevated levels of exogenous Fos protein.

Transcriptional repression mediated by the serum response factor.

The serum response element (SRE) contributes to transcriptional repression of the c-fos proto-oncogene. We show that the transcription factor SRF is able to repress SRE-dependent transcription, apparently by sequestering a co-activator. Only the DNA-binding core region is required for this SRE-dependent repression. Furthermore the phosphorylation status at potential casein kinase II sites within an N-terminal repression domain affects SRE-independent transcription. SRF may thus pleiotropically influence cellular transcription, representing a novel aspect of SRF function.

Regulatory squelching.

An important function of transcription factors may be to sequester coactivators or corepressors of transcription. In this manner transcription factors could regulate in trans the activity of promoters to which they do not bind. This may be of widespread significance as a mechanism to control cell cycle-dependent and differentiation-specific transcriptional activity within eukaryotic cells. Therefore squelching in vivo may be important than hitherto appreciated.

Fibroblasts at the transection site of the injured goldfish optic nerve and their potential role during retinal axonal regeneration.

The region at and around the site of optic nerve transection (ONS) in goldfish, topologically the equivalent of the glial scar in mammals, is reported to remain free of astrocytes over weeks, but its cellular constituents are unknown. To learn what type of cell occupies the site of injury and thus provides support for the rapidly regenerating retinal growth cones, immunostaining experiments at the light microscopic level and electron microscopic examinations were undertaken. Between 2 and 30 days after ONS, an area up to 150 micrograms wide at the transection site exhibits intense anti-fibronectin immunoreactivity. This site contained cells and processes with ultrastructural characteristics of fibroblasts and abundant collagen fibrils. Moreover, on fibroblast cultures derived from regenerating optic nerves, retinal axons grew to considerable density in vitro. Since fibroblasts are constituents of the interfascicular spaces and outer nerve sheath of the normal goldfish optic nerve, the present data imply that fibroblasts of either source migrate into the lesion. Judging form fibronectin immunostaining they remain there during the passage of regenerating axons, and thus may provide physical and perhaps molecular support for axon growth. The fibroblasts are again restricted to interfascicular spaces after restoration of the astrocytic glia limitans around regenerated fascicles.

Cholinergic motor control of sea urchin tube feet: evidence for chemical transmission without synapses.

Isolated tube feet of Strongylocentrotus franciscanus contract briefly when the outer epithelium is touched. Similar twitch-like contractions can be induced by electrical stimulation of the outer surface of the tube foot. These responses appear to be chemically mediated. The following evidence indicates that the transmitter substance may be acetylcholine (ACh): ACh causes muscle contraction. This effect and that of electrical stimuli is potentiated by anticholinesterase agents and is antagonized by cholinergic blocking agents. Anaesthesia with chloralhydrate or chloretone abolishes responsiveness to mechanical or electrical stimulation but not to ACh. Desensitization with carbachol prevents responses to ACh and to mechanical or electrical stimulation. There are no neuromuscular synapses and no axons can be detected which cross the connective tissue layer which separates the muscle fibres from the subepithelial nerve plexus. The latter is known to contain conspicuous amounts of ACh; nerve terminals containing clear vesicles invest the outer surface of the connective tissue layer. All evidence indicates that chemical transmission involves diffusion of ACh (released from activated nerve terminals) across this connective tissue layer which is around 5 micron thick in fully extended tube feet but may have a thickness of 20 or even 25 micron in less extended ones. Calculations based on equations describing transmitter diffusion prove the feasibility of such a mechanism.

Scanning electron microscopy of echinoid podia.

Tube feet of the sea urchin Strongylocentrotus franciscanus were studied with the scanning electron microscope (SEM). By use of fractured preparations it was possible to obtain views of all components of the layered tube-foot wall. The outer epithelium was found to bear tufts of cilia possibly belonging to sensory cells. The nerve plexus was clearly revealed as being composed of bundles of varicose axons. The basal lamina, which covers the outer and inner surfaces of the connective tissue layer, was found to be a mechanically resistant and elastic membrane. The connective tissue appears as dense bundles of (collagen) fibers. The luminal epithelium (coelothelium) is a single layer of flagellated collar cells. There is no indication that the muscle fibers, which insert on the inner basal lamina of the connective tissue layer are innervated by axons from the basi-epithelial nerve plexus. The results agree with previous conclusions concerning tube-foot structure based on transmission electron microscopy, and provide additional information, particularly with regard to the outer and inner epithelia.

The innervation pattern of crustacean skeletal muscle. Morphometry and ultrastructure of terminals and synapses.

The innervation pattern of distal muscle fibers of the opener muscle of walking legs of crayfish (Astacus leptodactylus) was investigated using methylene-blue staining, cobalt infiltration, and electron microscopy. A quantitative analysis of the entire innervation of single muscle fibers was attempted. It was found that instead of the generally assumed parallel array of numerous excitatory and inhibitory terminals, innervation consists of only a few branched terminals. The branches of excitatory and inhibitory terminals lie side-by-side. Both types are characterized by numerous varicosities (see Fig. 9B). The aggregate length of excitatory as well as inhibitory terminals on one muscle fiber is, on the average, about 1,500 micrometer with a total of 152 varicosities spaced about 10 micrometer apart. The average diameter of the varicosities is 4.26 micrometer, that of the connecting thin segments about 0.5 micrometer. Total terminal surface of motor or inhibitory terminals amounts to about 10,000 micrometers2 per muscle fiber. There are approximately 2,000 motor synapses on each muscle fiber, but their average total area is only about 6% of the terminal membrane area, or 0.06% of the (idealized) muscle fiber surface. There are conspicuous differences in the postsynaptic specializations associated with excitatory and inhibitory terminals; these are described in detail. The results are discussed in a functional context and with regard to design and results of electrophysiological experiments.

Ultrastructure of sea urchin tube feet. Evidence for connective tissue involvement in motor control.

An analysis of the ultrastructure of the tube feet of three species of sea urchins (Strongylocentrotus franciscanus, Arbacia lixula and Echinus esculentus) revealed that the smooth muscle, although known to be cholinoceptive, receives no motor innervation. The muscle fibers are attached to a double layer of circular and longitudinal connective tissue which surrounds the muscle layer and contains numerous bundles of collagen fibers. On its outside, the connective tissue cylinder is invested by a basal lamina of the outer epithelium to which numerous nerve terminals are attached. These are part of a nerve plexus which surrounds the connective tissue cylinder. The plexus itself is an extension of a longitudinal nerve that extends the whole length of the tube foot. It is composed of axons, but nerve cell bodies and synapses are conspicuously lacking, suggesting that the axons and terminals derive from cells of the radial nerve. Processes of the epithelial cells penetrate the nerve plexus and attach to the basal lamina. There is no evidence that the epithelial cells function as sensory cells. On the basis of supporting evidence it is suggested that the transmitter released by the nerve terminals diffuses to the muscle cells over a distance of several microns and in doing so affects the mechanical properties of the connective tissue.

Co-occurrence of CArG boxes and TCF sites within viral genomes.

The transcription factor SRF is involved in the transduction of extracellular signals into nuclear responses, often in conjunction with ternary complex factors (TCFs). Here we report the identification of CArG box SRF binding-sites, and neighboring TCF binding-sites, in viral genomes. SRF binds and recruits TCFs to CMV, RSV and HTLV-1 viral genomes. At least one of two specific CArG boxes occurred in cytomegaloviruses in the 5' proximal region of the major immediate early gene, one always accompanied by a TCF site. This conservation was striking since neither the flanking sequences nor the spacing to the CAP site were conserved. Thus the ubiquitous SRF and TCF molecules may control events in the life cycle of viruses.

Crosslinking of SRF to the c-fos SRE CArG box guanines using photo-active thioguanine oligodeoxynucleotides.

6-Thioguanine (thioG) was chemically incorporated into 25-base oligodeoxynucleotides encoding the c-fos serum response element (SRE) at positions corresponding to each guanine of the CArG box, which only slightly impaired DNA binding by the Serum Response Factor (SRF). Upon exposure to long wavelength UV light each thioG-containing SRE could be crosslinked to SRF, with efficiencies ranging from < 1 to 25% of the complex depending on the position of thioG in the SRE and on the UV source used. Crosslinking was strongest to the 3' side of the CArG box, and to the outer rather than the inner CArG box guanines, consistent with hydrogen bonds formed between SRF and the outer guanines in the crystal structure [Pellegrini et al., Nature 376, 490, 1995]. The crosslinked product was found to be chemically unstable. Possible mechanisms of crosslink formation are discussed.

Sensory and neurosecretory innervation of leech nephridia is accomplished by a single neurone containing FMRFamide.

The neural control of the excretory system of the medicinal leech Hirudo medicinalis has been characterized morphologically and chemically using light and electron microscopy, immunocytochemistry and biochemistry. Immunoreactivity against RFamide-like peptides revealed elaborate neuronal aborizations of a neurone in the nephridium, around the urinary bladder sphincter and in the central nervous system. The processes arose from the nephridial nerve cell (NNC), a previously identified receptor neurone. Using a combination of reverse-phase high pressure liquid chromatography, radioimmunoassay and subsequent Edman degradation and mass spectrometry, authentic FMRFamide has been identified as the major peptide of the NNC. Sensory and neurosecretory innervation of the nephridia is thus accomplished by a single neurone, which is thought to modulate nephridial performance.