Recent advances in breast cancer research: from genes to management.
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Adenovirus E1A Proteins↗
Biomedical subjects
R J Muschel
Publications and source records attributed to R J Muschel.
Inhibition of matrix metalloproteinase 9 expression by a ribozyme blocks metastasis in a rat sarcoma model system.
Matrix metalloproteinases (MMPs) have been implicated in tumor progression, but the exact roles that each member of this family may play in contributing to the behavior of malignant tumors are only beginning to be understood. MMP-9 (gelatinase B or the 92-kDa gelatinase/type IV collagenase) expression has been associated with metastasis in a variety of model systems including that of rat sarcomas generated by transformation of rat embryo cells with rasH and myc. To determine the effect that expression of MMP-9 has in this system, we inhibited the expression of MMP-9 using a hammerhead ribozyme. Introduction of an expression vector for a ribozyme directed against the rat MMP-9 mRNA sequence into a metastatic rat embryo cell line transformed by rasH and myc (2.10.10) that constitutively secretes MMP-9 resulted in the absence of detectable MMP-9 mRNA and loss of released 92-kDa gelatinase activity. These cells were no longer metastatic in a lung colonization assay but retained tumorigenicity. Introduction of an expression vector for a control hammerhead ribozyme had no effect. These data document the requirement for MMP-9 expression in metastasis in this system.
Animals↗
Delayed cyclin B1 expression during the G2 arrest following DNA damage.
Exposure of cells to DNA damaging agents results in a G2 arrest. Exposure of HeLa cells to camptothecin, etoposide or nitrogen mustard for 1 h in S phase resulted in delayed expression of cyclin B1 mRNA during the G2 arrest. Initially the levels of cyclin B1 protein were low as well; however, with extended time the cells blocked in G2 regained higher levels of cyclin B1 protein. In the case of cells treated with nitrogen mustard the higher levels coincided with cells exiting the G2 block into G1. However, with camptothecin or etoposide treatment, while the accumulation of cyclin B1 protein was delayed, its levels eventually surpassed peak levels seen in control cells, in spite of the fact that cells were still blocked in G2. These cells did not continue to progress through the cell cycle indicating further complexity to the mechanisms underlying the G2 block. Decreased transcription and stability of cyclin B1 mRNA were shown to occur after treatment with these DNA damaging agents. These results indicate that suppression of cyclin B1 mRNA expression is one consequence of DNA damage in HeLa cells.
Antineoplastic Agents↗
The farnesyltransferase inhibitor FTI-277 radiosensitizes H-ras-transformed rat embryo fibroblasts.
Many tumor cells have a greater resistance to ionizing radiation than their normal counterparts, suggesting that the development of drugs that can reduce that radioresistance would potentiate the efficacy of radiation therapy. Because activated H-ras expression has been shown to markedly increase radiation resistance in some transformed cells, the inactivation of H-ras would then be predicted to radiosensitize these tumor cells, while leaving normal cells unaffected. H-ras depends for activity upon farnesylation, which can be blocked by farnesylation inhibitors, including the compound FTI-277. In keeping with this prediction, inhibition of H-ras processing using FTI-277 resulted in higher levels of apoptosis after irradiation and increased radiosensitivity in H-ras-transformed rat embryo cells but did not affect control cells. These experiments suggest that farnesylation inhibitors may prove clinically useful as radiosensitizers of tumors that depend on ras function.
Alkyl and Aryl Transferases↗
Regulation of radiation-induced apoptosis in oncogene-transfected fibroblasts: influence of H-ras on the G2 delay.
Primary fibroblasts, after serum withdrawal or after irradiation, do not undergo apoptosis. Myc-transfected fibroblasts, in contrast, undergo apoptosis upon serum withdrawal and after irradiation. We have studied the relationship of apoptosis induction to effects on the G2 phase cell cycle in a series of rat embryo cells transformed by rasH plus myc or immortalized by myc alone. In this system, while the presence of rasH had little effect on the extent of apoptosis induction by serum withdrawal, rasH greatly suppressed the apoptotic response of myc-transfected cells to X-rays. The cells into which rasH had been introduced showed a profound G2 arrest associated with suppression of cyclin B1 mRNA expression. In contrast, cells with myc alone had a minimal G2 delay after irradiation and no suppression of cyclin B1 mRNA expression. We hypothesize that rasH, by influencing the G2 response of cells to X-rays, exerts an anti-apoptotic effect. In support of this hypothesis; we found that treatment of cells with caffeine, an agent that relieves the G2 delay after irradiation resulted in increased apoptosis in the irradiated cells, but not in control cells.
Animals↗
Alterations in cell cycle control during tumor progression: effects on apoptosis and the response to therapeutic agents.
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Antineoplastic Agents↗
Induction of matrix metalloproteinase 9 expression in breast carcinoma cells by a soluble factor from fibroblasts.
Tumor-stromal interactions appear to play an important role in the induction of metalloproteinase expression in malignant tumors. We describe a tissue culture system in which expression of MMP-9 (gelatinase B or the 92 kDa type IV collagenase/gelatinase) was induced by co-cultivation of fibroblasts with breast cancer cell lines. While neither the breast cancer cells nor the normal rat embryo fibroblasts made MMP-9 alone in culture, human MMP-9 was made in the co-cultures. The MMP-9 was secreted in a latent form. The induction occurred at least in part through increases in the MMP-9 mRNA levels in the breast cancer cells. These increases did not appear to require protein synthesis. Conditioned medium from the fibroblasts could duplicate the induction of MMP-9 in the breast cancer cell lines. The active factor in the medium was inactivated by heat or by trypsin suggesting that it was a protein. This protein was in the size range of 30-100 kDa. Thus, fibroblasts could secrete a factor which was able to regulate the expression of MMP-9 in breast cancer cells.
Animals↗
Reducing the radiation-induced G2 delay causes HeLa cells to undergo apoptosis instead of mitotic death.
Cells exposed to radiation may undergo death through apoptosis or mitotic death. HeLa cells predominantly undergo mitotic death after irradiation. Treatment of these cells with caffeine has been shown to shorten the G2 delay after irradiation, and to decrease their survival. The kinase inhibitor staurosporine also decreases the radiation-induced G2 delay in HeLa cells. Here we extend these findings to show that the decrease in radiation-induced G2 delay mediated by caffeine or staurosporine is accompanied by a shift in the pathway of cell death from mitotic death to apoptotic death. The increase in apoptosis is further accompanied by decreased clonogenic survival after irradiation. Based on these findings we propose the hypothesis that one mechanism of enhancing cell killing by radiation is to trigger apoptosis by decreasing the G2 delay induced by irradiation.
Alkaloids↗
Link between tumor progression, proteases, and cell motility. Pathology B workshop.
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Animals↗
Cell cycle-dependent regulation of the cyclin B1 promoter.
Cyclin B1 mRNA expression varies through the cell cycle with its peak in G2/M. In cycling mammalian cells, its lowest level is in G1 with a steady increase in S until a level 50-fold greater than that in G1 is reached. In order to characterize the transcriptional component to this variation in expression, we cloned the upstream region 872 base pairs upstream from the start site of the cyclin B1 gene and have demonstrated that it confers cell cycle-dependent regulation onto two reporter genes, both chloramphenicol acetyltransferase and luciferase. Its activity was 25-fold greater in G2/M than in G1 in HeLa cells with intermediate activity in S. This cyclical activity could be seen with sequences encompassing only 90 base pairs upstream from the start site. Protein binding to this region was demonstrated using electrophoretic mobility shift assays, and the binding profiles appeared to vary depending upon the phase of the cycle in which the extracts are made. Thus, transcriptional control plays an important role in determining cyclin B1 mRNA levels, and cell cycle-dependent activity is regulated through interactions with the region 90 bases upstream from the start site.
Base Sequence↗
MMP-9 (gelatinase B) mRNA is expressed during mouse neurogenesis and may be associated with vascularization.
Expression of MMP-9 mRNA, a type IV collagenase gene product, was followed during embryonic development of the mouse brain using in situ hybridization. Murine embryos from 7.5 to 15 days after fertilization were sectioned and evaluated for MMP-9 expression. During early development, from day 7.5 to day 9, no signal was detected in the cells of the neuroepithelium or in cells of the cephalic mesenchyme of the neural tube. At day 11, gene expression was localized to the Rathke's pouch and the germinal zone of the primitive ventricular system. At day 13, but most notably at day 15, high levels of MMP-9 were expressed by progenitor cells in close association with the development of structures, such as the hypophysis, the choroid plexus, the ganglion cell layer of the retina and the uveal tract. High MMP-9 mRNA levels were also associated with dense cellular aggregates destined to form the highly vascular grey matter of the brain. The presence of MMP-9 mRNA was confirmed using a ribonuclease protection assay. A 105 kDa gelatinase, consistent with the expected molecular mass for the murine MMP-9, was detected in embryonic brain extracts by substrate gel electrophoresis. To our knowledge, this is the first report on the localization of MMP-9 in developing neural tissues. Our results suggest that MMP-9 expression may have a previously unsuspected role in neural development.
Animals↗
The effect of E1A transfection on MMP-9 expression and metastatic potential.
The expression of MMP-9 in rat embryo fibroblasts (REF) transformed with Ha-ras or with Ha-ras and v-myc is associated with metastatic behavior. In contrast, REF transformed with Ha-ras and the adenovirus E1A genes (E1A) are tumorigenic, do not release MMP-9 and are rarely metastatic. In this report, we establish that E1A expression results in decreased levels of MMP-9 mRNA in an Ha-ras and v-myc transformed cell line and examine which of the functional domains of E1A participate in the inhibition of MMP-9 expression and which contribute to the suppression of metastasis. The metastatic 2.10 REF line, derived by co-transfection with v-myc and Ha-ras, constitutively expresses high levels of MMP-9 (92 kDa gelatinase). Transfection of E1A wild-type plasmids into this cell line eliminates detectable MMP-9 mRNA expression and greatly reduces MMP-9 activity. Transfection of 2.10 with E1A plasmids encoding mutations in conserved region 2 (CR2) retained inhibition of MMP-9 similar to the inhibition seen with wild-type E1A. Transfection with E1A containing mutations in CRI or the amino terminal region diminished, but did not fully inhibit, MMP-9 expression. In contrast, inhibition of MMP-9 was lost in with E1A mutations in CR3. Cells transfected with E1A mutants in CR1, the amino terminal region or CR3 retained metastatic behavior. Our findings delineate the regions of E1A responsible for MMP-9 inhibition and further define the domains of E1A responsible for inhibition of metastasis.
Adenovirus E1A Proteins↗
Induction of apoptosis at different oxygen tensions: evidence that oxygen radicals do not mediate apoptotic signaling.
Apoptosis has been hypothesized to be mediated through the induction of free radicals via oxidative pathways. Furthermore, it has been proposed that Bcl-2 acts to inhibit apoptosis induced by a wide variety of stimuli by preventing the production of oxygen-derived free radicals. Since the generation of oxygen free radicals is dependent upon oxygen concentration, this hypothesis would lead to the prediction that the concentration of oxygen should affect the induction of apoptosis. In order to test this prediction, we have examined the induction of apoptosis in T-lymphoma cell lines S49.1 and WEHI 7.1 by dexamethasone and by withdrawal of serum from myc-immortalized fibroblasts in 95% oxygen, atmospheric oxygen (20%), and hypoxic conditions of up to 125-fold less oxygen. Culture in 95% oxygen induced apoptosis in all cells tested, confirming that oxidative damage can lead to apoptosis. However, for one cell line, WEHI 7.1, hypoxia also induced apoptosis. Furthermore, for the other cell lines tested, induction of apoptosis by either dexamethasone or by serum withdrawal was not affected by hypoxia. These results are not consistent with the hypothesis that apoptosis is mediated via oxygen-generated free radical formation.
Animals↗
Evidence for post-transcriptional regulation of cyclin B1 mRNA in the cell cycle and following irradiation in HeLa cells.
Cyclin B1 mRNA expression varies markedly through the cell cycle with its peak in G2/M and lowest level in G1. Cyclin B1 mRNA levels are also transiently reduced in HeLa cells after gamma-irradiation, coincident with the radiation-induced G2 block. In order to understand the mechanisms underlying these variations, we have measured cyclin B1 mRNA stability in HeLa cells during different phases of the cell cycle. The half-life of the mRNA measured after actinomycin D administration is 1.1-1.8 h in both early and late G1, 8 h in S and 13 h in G2/M. We therefore conclude that altered RNA stability is important in modulating cyclin B1 mRNA levels through the HeLa cell cycle. Furthermore, 3 h after irradiation of HeLa cells in S phase with 10 Gy, the half-life of cyclin B1 mRNA is reduced to 5 h; it is further reduced to 2-3 h at 14 h after irradiation. Thus, decreased stability contributes to the reduction in cyclin B1 mRNA following irradiation.
Blotting, Northern↗
Developmental expression of MMP-9 (gelatinase B) mRNA in mouse embryos.
Considerable remodeling of the extracellular matrix as well as cellular migration takes place during embryogenesis. Since the metalloproteinase MMP-9 is implicated in these functions in cancer cells, we studied the patterns of expression of MMP-9 mRNA during the development of post-implantation mouse embryos. MMP-9 mRNA was detected using the ribonuclease protection assay in poly A+RNA from 13 to 17 day embryos, but not at 11 days. In order to localize these transcripts, in situ hybridization was performed on sections of murine embryos from 7.5 to 15 days of gestation. At the time of implantation, MMP-9 mRNA was localized to the invading trophoblast cells. Strong signals were also seen in the yolk sac. No signal for MMP-9 mRNA was seen by in situ hybridization in the embryo until day 11 when detectable reaction was seen in the central nervous system. By day 15 strong signals were seen in the liver, in the developing bronchial epithelium of the lungs and in the primordial alveoli, in the epithelium of the thyroid gland, in the thymus, in the endochondrial plates of the bone, and in neural cells. The liver from day 15 embryos contained gelatinase activity at 105 kDa consistent with MMP-9. Thus, MMP-9 expression appears to be expressed in specific organs in a precise temporal sequence during development.
Animals↗
Effects of ionizing radiation on cell cycle progression. A review.
Irradiation of normal eukaryotic cells results in delayed progression through the G1, S, and G2 phases of the cell cycle. The G1 arrest is regulated by the p53 tumor suppressor gene product. Irradiation results in increased expression of p53, which in turn induces a 21 kDa protein, WAF1/Cip 1, that inhibits cyclin CDK kinases. S-phase delay is observed after relatively high doses of radiation. This delay has both radiosensitive and radioresistant components, corresponding to inhibition of DNA replicon initiation and DNA chain elongation, respectively. The mechanism for this delay is as yet undefined, but the extent of the delay appears to be under genetic control and is sensitive to the kinase inhibitor staurosporine. A delay in G2 has been demonstrated in virtually all eukaryotic cells examined in response to irradiation. Our studies have focused on the mechanisms responsible for this delay. Cyclin B1 and p34cdc2 are cell cycle control proteins that together form a kinase complex required for passage through G2 and mitosis [22]. Control of radiation-induced G2 delay is likely therefore to involve modulation of cyclin B1/p34cdc2 activity. We have shown in HeLa cells that cyclin B1 expression is decreased in a dose-dependent manner following irradiation. This decrease is controlled at both the level of mRNA and protein accumulation. We have also shown that radiation-sensitive rat embryo fibroblast lines (REF) immortalized with v- or c-myc display a minimal G2 delay when compared to radiation resistant cells transformed with v-myc + H-ras. These REF lines respond to irradiation with a decrease in cyclin B mRNA, which parallels the extent of their respective G2 delays.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
92-kd type IV collagenase (matrix metalloproteinase-9) activity in human amniochorion increases with labor.
To determine whether specific collagenolytic enzymes are expressed in human fetal membranes with labor, we examined gelatinase activity in extracts of amniochorion by zymography. The 92-kd gelatinase (MMP-9) was barely detectable in extracts of fetal membranes before the onset of labor but was readily demonstrable in extracts prepared from membranes isolated from laboring women or membranes collected immediately after delivery. In contrast, the 72-kd gelatinase (MMP-2) was detectable in extracts from pre- and post-labor membranes. Ethylenediaminetetracetic acid and the tissue inhibitor of metalloproteinases, TIMP-1, inhibited the gelatinase activities detected by zymography, confirming that the enzymes are metalloproteinase. Assay of amniochorion gelatinase activity using a radiolabeled denatured collagen substrate revealed a more than twofold increase in activity comparing pre-labor with post-labor fetal membrane extracts. A function-blocking anti-MMP-9 monoclonal antibody inhibited pre-labor membrane gelatinase activity by approximately 11.5%, which was only slightly greater inhibition than observed with irrelevant monoclonal antibodies. However, post-labor membrane gelatinase activity was reduced by 53% by the function-blocking antibody, indicating that MMP-9 is a major contributor to the increased gelatinase activity extractable from post-labor membranes. Western blot analyses demonstrated increased MMP-9 protein in amniochorion extracts after onset of labor. MMP-9 protein and mRNA were co-localized in amnion epithelium, underlying macrophages and chorion laeve trophoblast and decidual cells after labor. We conclude that 1) MMP-9 activity and protein in human amniochorion increases with labor and 2) MMP-9 is expressed by amnion epithelium, macrophages and chorion laeve trophoblast and decidual cells. The increased expression of MMP-9 may result in degradation of the extracellular matrix of the fetal membranes and facilitate their rupture under both physiological and pathological conditions.
Amino Acid Sequence↗
Alternate polyadenylation in rodent cells results in two differentially expressed cyclin B1 mRNAs.
Cyclin B1 is required for the G2/M transition. In HeLa cells, the level of both its protein and mRNA varies dramatically through the cell cycle. In this study we examine the expression of cyclin B1 mRNA in Chinese hamster ovary cells and 3.7, a transformed rat embryo fibroblast line. In contrast to HeLa cells in which a single transcript is seen, both of these lines express two distinct transcripts of approximately 1.4 and 2.7 kbp in length which appear to be made using different polyadenylation sites. In CHO cells, the level of the upper band varies modestly through the cell cycle while the lower band remains almost constant. In contrast to HeLa cells, CHO cells in G1 express a significant amount of cyclin B1 mRNA. In synchronized 3.7 cells, the level of both transcripts is very low in G1 and rises in parallel as cells progress through S and G2, but the longer transcript disappears faster as cells reenter G1.
Animals↗