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The influence of activated ras oncogenes on the sensitivity of human tumor cells to killing by radiation has been an unresolved question in radiobiology. We have examined this question by measuring the radiation sensitivity of human tumor cell lines with oncogenic mutations in their H- or K-ras genes after treatment with prenyltransferase inhibitors that prevent the posttranslational modification of ras required for its activity. Using two measures of clonogenic survival, we have demonstrated radiosensitization in cell lines with oncogenic H-ras mutations or with oncogenic K-ras mutations when ras processing was inhibited by prenyltransferase inhibitor treatment. In contrast, the inhibition of ras processing in cell lines expressing wild-type ras had no effect on radiation-induced cell death. The prenyltransferase inhibitors themselves inhibited clonogenic survival in some cases, but this inhibition did not correlate with ras mutational status. Although treatment with prenyltransferase inhibitors and radiation resulted in a greater reduction of clonogenicity than either treatment alone in cells with wild-type ras, treatment with both agents had a synergistic effect on cell killing in tumor cells with ras mutations. Our results demonstrate that the inhibition of oncogenic ras activity in human tumor cells can reduce the radiation survival of these cells, suggesting that oncogenic ras can contribute to radiation resistance in human tumors. These results further demonstrate the potential of using prenyltransferase inhibitors in combination with radiotherapy in the treatment of human malignancies.

Several important and possibly interrelated functions have been identified for the HIV-1 accessory gene product Vpr. These include import of the HIV reverse transcription complex into the nucleus of nondividing cells, cellular differentiation including cell cycle arrest at the G2/M phase border, immune suppression, and enhancement of virus replication. We have cloned a candidate Vpr ligand, termed human Vpr interacting protein (hVIP/MOV34), by using a yeast two-hybrid assay. This gene is homologous to a simultaneously identified 34-kDa human mov34 homologue. The MOV34 family includes proteins that function as transcriptional and proteolytic regulators of cell growth and differentiation. We demonstrate direct interactions between the putative ligand hVIP/MOV34 and Vpr in vitro and in vivo. hVIP/MOV34 localizes to the nucleus and appears to function as a component of the cell cycle cascade. We observe an association between the induction of cell cycle arrest at the G2/M phase border by Vpr and a change in the subcellular localization of hVIP/MOV34 from a nuclear to a perinuclear localization. This was further associated with the inhibition of maturation promoting factor-associated histone H1 kinase activity. We conclude that hVIP/MOV34 is involved in the regulation of the cell cycle and a likely cellular cofactor for HIV-1 Vpr.

The 92-kDa type IV collagenase (MMP-9) is a metalloproteinase frequently localized in both tumor stroma and in tumor cells, particularly at the tumor invasion front. To explore the factors regulating transcriptional activation of MMP-9 in stromal cells, we used a model system in which fibroblast MMP-9 expression can be upregulated by cell-cell contact with metastatic transformed rat embryo cells. Using transient transfection of reporter gene constructs containing 5'-deleted or mutated MMP-9 promoter fragments, as well as electrophoretic mobility shift assays, the upstream NFkappaB, SP-1, and Ets sites and the downstream AP-1 site and retinoblastoma binding element were shown to be necessary for basal transcriptional activity of fibroblast MMP-9. In contrast only Ets or SP-1 appeared to be involved in contact-mediated induction of MMP-9. Mutation of the upstream AP-1 site increased both basal and contact-stimulated promoter activation. Deletion of the alternating purine-pyrimidine repeat in the downstream promoter decreased transcriptional activity. Together these findings suggest that Ets and SP-1 are the central transcriptional activators of MMP-9 gene expression in fibroblasts specifically responding to tumor cell contact, and that promoter conformation may regulate MMP-9 expression.

Although a number of effective therapies are available for localized prostate cancer, metastatic prostate cancer is difficult to treat and impossible to cure. Identification of the gene products that enable a prostatic carcinoma cell to metastasize should facilitate an understanding of the processes leading to metastasis. To characterize the contribution of matrix metalloproteinase-9 (MMP-9, gelatinase B or the 92-kd type IV gelatinase/collagenase) to the development of metastasis in prostate cancer, we reduced MMP-9 expression in metastatic murine prostatic carcinoma cells using a ribozyme. The ribozyme transfected cells had lower basal levels of MMP-9 as well as decreased levels after stimulation by transforming growth factor-beta or phorbol 12-myristate 13-acetate when compared with the parental cells or with control transfectants. The cells with down-regulated MMP-9 were unable to form lung colonies in the experimental metastasis assay, whereas the controls and parental cells readily formed metastases. All cell types readily formed tumors after injection and down-regulation of MMP-9 did not adversely affect the rate of tumor growth. Thus, MMP-9 expression is required for hematogenous metastasis in a murine prostate model system raising the possibility that it may play an equivalent role in human prostate cancer.

The 92 kd type IV collagenase/gelatinase (MMP-9) is important in mediating basement membrane and extracellular matrix degradation in metastasis. Because MMP-9 is made in tumor cells, but not in quiescent normal cells, we wished to identify the transcriptional elements responsible for its synthesis in tumor cells. We chose to characterize transcriptional regulation of the MMP-9 gene in a highly metastatic H-ras and v-myc transformed rat embryo cell line which overexpresses MMP-9. Using transient transfection of reporter gene constructs containing either 5'-deleted or mutated MMP-9 promoter fragments, as well as electrophoretic mobility shift assays, we have demonstrated that multiple transcription factor consensus binding motifs in the promoter, including those for NFkappaB, SP-1, Ets, AP-1, and a retinoblastoma binding element, participate in transcriptional regulation of MMP-9 expression in this cell line. Also, deletion of an alternating purine-pyrimidine tract in the downstream promoter was found to decrease transcriptional activity, suggesting that promoter conformation may be important in MMP-9 regulation. Thus multiple pathways leading to activation of NFkappaB, SP-1, Ets, AP-1, and retinoblastoma binding factors in tumor cells all may contribute to MMP-9 transcription and hence to metastasis.

Progression through the cell cycle in somatic eukaryotic cells is regulated by variations in the levels of cyclin proteins. These protein levels are in turn regulated by cyclical oscillations in their mRNA levels. We show here that regulation of RNA stability plays a role in the mechanisms underlying cell cycle progression. Both cyclin A and B1 messages are expressed at high levels in G2-M and at low levels in early G1. The half-lives of their messages mirror this pattern, long in G2-M (>8 h) and short in early G1 (1-2 h). However, there is evidence of specificity to these changes, because the cyclin A message becomes stable at the G1-S boundary, whereas the cyclin B1 message is unstable until later in S phase. Furthermore, although cyclin B1 mRNA levels are lowered after irradiation because of enhanced instability, cyclin A mRNA levels and message stability are unaffected by irradiation. Additional evidence of specificity was found in an analysis of cyclin E mRNA stability, which remains constant through the cell cycle, although the cyclin E message displays cell cycle-dependent changes in expression. These studies suggest that specific alterations in RNA stability are an important component in regulating the expression of cyclins A and B1 and hence in controlling the cell cycle.

Irradiation of tumor cells results in a G2 delay, which has been postulated to allow DNA repair and cell survival. The G2 delay after irradiation is marked in HeLa and other cells by delayed expression of cyclin B1. To test whether this depression of cyclin B1 contributes to the G2 delay, we induced cyclin B1 expression in irradiated HeLa cells using a dexamethasone-inducible promoter. Induction of cyclin B1 after radiation abrogated the G2 delay by approximately doubling the rate at which the cells reentered mitosis, whereas dexamethasone itself had no effect. However, overexpression of cyclin B1 did not eliminate the G2 delay in irradiated cells. In unirradiated cells, overexpression of cyclin B1 had no effect on cell cycle progression. Confirmation that reduction of cyclin B1 levels would prolong G2 was provided using antisense oligonucleotides to cyclin B1. These results demonstrate that cyclin B1 levels control the length of the G2 delay following irradiation in HeLa cells but do not exclude additional mechanisms controlling the mitotic delay after irradiation.

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.

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.

Hydroperoxides are reduced in mammalian cells by a coupled enzyme pathway involving glutathione peroxidase, glutathione reductase and the oxidative limb of the pentose cycle. Oxidation of glucose-6-phosphate by the pentose cycle yields two molecules of NADPH, which can reduce two hydroperoxide molecules to the corresponding alcohol. Rat embryo fibroblasts (REF) transfected with v-myc reduce hydroperoxides slower than the primary REF cell line-measured both as real time peroxide loss and as increased glucose oxidation via the pentose cycle. The v-myc transfected cell line is 50-fold more sensitive to the toxic effects of tBu-OOH. The decreased reduction of peroxides by v-myc transfected cells is not due to changes in the activities of GSH reductase or the enzymes of the oxidative pentose cycle, since diamide stimulates PC activity equally in both cell lines. In addition, the activities of these enzymes, measured in cell homogenates do not differ significantly between the cell lines. Also total GSH peroxidase activity, assayed in cell homogenates, is not significantly different between the cell lines. Two human tumour cell lines which overexpress myc family proteins: NCI-H69, a small-cell lung cancer line which expresses elevated levels of N-myc, and HL-60 cells which overexpress c-myc, also exhibit low levels of pentose cycle stimulation in the presence of tBu-OOH, and a decreased capacity to reduce hydrogen peroxide by peroxide electrode.

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.

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.

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.

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.

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.

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.

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)

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.

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.

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.

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.

The irradiation of cells results in delayed progression through the G2 phase of the cell cycle. Treatment of irradiated HeLa cells with caffeine greatly reduces the G2-phase delay, while caffeine does not alter progression of cells through the cell cycle in unirradiated cells. In this report we demonstrate that treatment of HeLa cells with the kinase inhibitor staurosporine, but not with the inhibitor H7, also results in a reduction of the G2-phase arrest after irradiation. Cell cycle progression in unirradiated cells is unaffected by 4.4 nM (2 ng/ml) staurosporine, which releases the radiation-induced G2-phase arrest. In HeLa cells, the G2-phase delay after irradiation in S phase is accompanied by decreased expression of cyclin B1 mRNA. Coincident with the reduction in G2-phase delay, we observed an increase in cyclin B1 mRNA accumulation in irradiated, staurosporine-treated cells compared to cells treated with irradiation alone. Caffeine treatment of irradiated HeLa cells also resulted in an elevation in the levels of cyclin B1 message. These results support the hypothesis that diminished cyclin B1 mRNA levels influence G2-phase arrest to some degree. The findings that both staurosporine and caffeine treatments reverse the depression in cyclin B1 expression suggest that these two compounds may act on a common pathway of cell cycle control in response to radiation injury.

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.

Many studies have demonstrated the effect of oncogene transfection on the radiation sensitivity of primary rat embryo fibroblasts. Our results indicate that transformation by H-ras plus v-myc oncogenes confers radiation resistance to a much greater extent than transformation by either gene alone. We have further shown that the radioresistant phenotype is accompanied by a prolonged G2-phase delay. This is consistent with the hypothesis that the extent of this delay is an important determinant of radiation sensitivity. The study of cyclin expression during the progression of cells through G2 and M phase after irradiation has also revealed several possible mechanisms for induction of G2-phase arrest. Control of cyclin B levels was seen both at the mRNA level as evidenced by decreased cyclin B mRNA expression after irradiation in the S phase, and at the protein level as demonstrated after irradiation in G2 phase. It remains to be determined how these mechanisms might be differentially regulated in radioresistant and sensitive cells.

We have used in situ hybridization for RNA to localize cells containing mRNA for the 92 kd gelatinase in carcinomas of the lung. We used archival material to analyze sections from 12 cases of squamous cell carcinomas of the lung including six stage I and three stage II and from three cases of adenocarcinoma of the lung. Presence of mRNA in the tissue was verified by in situ hybridization for gamma actin. The 92 kd gelatinase mRNA was found in all 12 squamous cell carcinomas tumors and was highly expressed in the tumor cells themselves. In addition, it was found in host stromal cells surrounding the tumor, but not in normal lung fibroblasts. In contrast it was not found in the adenocarcinomas of the lung or in the stroma surrounding these tumors. The mRNA for the 92 kd gelatinase was present in normal pulmonary tissue, bronchial epithelium, basal cell hyperplasia of bronchial epithelium, alveolar macrophages, and focally in bronchial mucous glands. It was not present in normal alveoli, vascular cells, cartilage, or most lymphocytes. We corroborated the presence of the mRNA for the 92 kd gelatinase by ribonuclease protection assay. The levels of mRNA for the 92 kd gelatinase in two specimens of squamous cell carcinoma were 6- to 10-fold greater than in the nonneoplastic tissue and two adenocarcinoma specimens.

Many enzymes capable of proteolytic degradation of extracellular matrix and basement membranes have been implicated in tumor progression, including the matrix metalloproteinases, cathepsins, plasminogen activators, and heparanase. Matrix metalloproteinases, a family of zinc-dependent proteases, participate in several steps in tumor progression, including invasion, metastasis, and angiogenesis. In this review, we will give a brief overview of this protease family, and we will review in vitro and in vivo evidence implicating a particular metalloproteinase, the 92-kD type IV collagenase/gelatinase (MMP-9 or gelatinase B), as well as other metalloproteinases, in cancer progression. Finally, using recent studies from our laboratory, we will demonstrate the importance of both tumor cell and host stromal cell production of MMP-9 in tumor progression.

Purpose: To evaluate the hypothesis that the radiation induced G2 delay in the cell cycle is associated with radiation induced effects on cyclin B expression in a rodent cell system., Methods and Materials: Two rodent, rat and Chinese hamster, cyclin B cDNAs were cloned and characterized. The two rodent species were 85% and 86% identical, respectively, when compared to the human cyclin B, indicating that they are the rodent homologous of cyclin B. 3.7 cells (rat embryo cells transformed by H-ras and v-myc) were synchronized and then irradiated. Flow cytometry and Northern blots were performed to evaluate the effects of radiation on cyclin expression in relation to phase of the cell cycle., Results: Examination of the rodent cyclin B sequences revealed only two regions with significant divergence to the human sequence, one in the lysine rich region adjacent to the cyclin destruction box, which is the putative site for ubiquitination, and one at the C terminal end. Although many of the amino acids diverged in the lysine rich region, the positions of the lysines themselves were virtually invariant suggesting their potential importance in ubiquitination. Both rodent species were also noted to have a PEST-like sequence which occurs in the human, but not in non-mammalian cyclins cloned to date and could also potentially contribute to rapid destruction. The rat and Chinese hamster mRNAs contain much longer 3' untranslated regions than the published human sequence with multiple AUUUA and AUUU motifs which are seen in other mRNAs with rapid turnover times. This feature has not been previously found in cyclin mRNAs. In addition we have found that in the 3' region of the rodent cDNAs we find two potential polyadenylation sites suggesting that this gene may have several transcripts. Our studies suggest that multiple mechanisms of control of mammalian cyclin B destruction exist, both at the mRNA and protein level. Evidence is also provided that the levels of rat cyclin B mRNA peaks during G2/M. Irradiation is shown to induce a G2 delay in synchronized 3.7 cells, compared to unirradiated controls, and the delay is temporally related to decreased levels of cyclin B mRNA expression. Since the G2 delay induced by ionizing radiation may contribute to the ability of cells to survive irradiation, cyclin B expression may be a key component in the determination of sensitivity or resistance to radiation therapy., Conclusion: The isolation and characterization of two rodent cyclin B's confirm that multiple mechanisms of control of mammalian cyclin B destruction exist. Our studies show that rat cyclin B expression is influenced by radiation and is temporally related to the delay in the G2 phase induced by radiation.

Ionizing radiation induces a G2 delay in eukaryotic cells. Since mitotic cyclins are required to trigger the transition from G2 into and through mitosis, we chose to investigate their expression after irradiation in HeLa cells. In normally cycling HeLa cells, both cyclin A and B mRNA and protein levels rise dramatically in G2/M and rapidly fall coincident with the completion of mitosis. The rise of cyclin A mRNA at the S/G2 boundary slightly precedes that of cyclin B mRNA. Although the peaks of expression of each of these molecules overlap, cyclin A mRNA and protein diminish before cyclin B. After irradiation in S, cyclin A mRNA and protein levels rose with the same kinetics as in the controls, but ultimately exceeded the levels seen in the control population. Cyclin A mRNA and protein levels remained high throughout the G2 delay induced by irradiation. In contrast, cyclin B mRNA and protein levels did not rise as the irradiated cells entered G2/M. Only later, before the irradiated cells exited from G2/M, did levels of cyclin B reach the levels seen in the unirradiated controls. The decreased amount of cyclin B mRNA and protein was inversely proportional to the dose of radiation. These data indicate that irradiation that results in a G2 delay appears to block cells at a point after production of cyclin A but before cyclin B can be fully expressed and that cells do not exit from the delay until cyclin B is again expressed. Thus, cyclin A and cyclin B expression respond differentially to radiation, with cyclin A rising at the same time as the control and to even higher levels than that seen in the controls, whereas cyclin B shows a temporal delay in expression.

The levels of cyclin B mRNA and protein rise rapidly in G2 + M phase, and fall at the end of mitosis. The studies described here were initiated to determine the effects of ionizing radiation on the level of cyclin B bearing in mind that the division delay induced by ionizing radiation might be influenced by the expression of cyclin B. After irradiation in S phase, the cyclin B mRNA in HeLa cells was measured as the cells proceeded through the cell cycle. Instead of the usual rise, after irradiation cyclin B mRNA levels remained low during the G2 delay. After irradiation in G2 phase, cyclin B mRNA was readily detectable although at slightly lower levels than in the controls. However, cyclin B protein was markedly decreased in amount.

Cathepsins B and L and their endogenous inhibitors were evaluated in rat embryo fibroblast lines which have been developed as a model system for the study of malignant progression and metastatic capability. Three groups of lines were analyzed: 1) immortalized/non-tumorigenic, 2) tumorigenic/metastatic lines transfected with c-Ha-ras, and 3) metastatic revertants transfected with c-Ha-ras+the E1A region of adenovirus type 2. The metastatic revertants are tumorigenic, but non-metastatic. No correlation was seen between tumorigenicity and metastatic potential and the level of expression of cathepsin B or the subcellular distribution of cathepsins B and L. However, cathepsin L activity was increased 2-fold in the 4R metastatic line. Although transfection of aneuploid 3T3 fibroblasts with ras has been shown to increase the expression of cathepsin L and cathepsin B, transfection of the diploid rat embryo fibroblasts with ras did not correlate with increased expression of cathepsin L or cathepsin B. However, ras transfection of the rat embryo fibroblasts was associated with a significant (4-15-fold) decrease in the activity of heat-stable cysteine endopeptidase inhibitors. Thus, in tumorigenic rat embryo fibroblast lines, regulation of the activities of cysteine endopeptidases by their endogenous inhibitors may be compromised, resulting in increased effective activities of the cysteine endopeptidases.

After exposure to ionizing radiation, eukaryotic cells undergo a division delay which is reflected by increased time spent in the G2 portion of the cell cycle. Recent information identifies increased levels of mitotic cyclins as key biochemical events initiating mitosis. In HeLa cells cyclin B mRNA and protein levels have been shown to increase in G2 and to decrease after division is completed. Cyclin B protein binds to cdc2, resulting in histone kinase activity which is necessary for the initiation of mitosis. Accordingly, we chose to investigate how cyclin B mRNA and protein levels were perturbed by irradiation in order to gain further understanding of the mechanisms by which ionizing radiation leads to a division delay. Our experiments revealed at least two effects on cyclin B regulation which might contribute to the division delay: (a) when HeLa cells were irradiated in S phase, there was a delay in the accumulation of cyclin B mRNA; (b) when cells were radiated in G2 phase, at a time when mRNA levels were increasing, a division delay was induced which coincided with a markedly lowered level of cyclin B protein despite high levels of the mRNA.

Cell cycle perturbation after irradiation was studied in five cell lines transfected with oncogenes. Two immortalized, radio-sensitive cell lines with D0s of 1.06 and 1.08 Gy were compared to three radioresistant cell lines with D0s of 1.68-2.17 Gy. The sensitive cell lines were transfected with the v-myc or c-myc oncogenes, the resistant cell lines with the v-myc plus H-ras oncogenes. Exponentially growing populations were exposed to 5, 10, or 15 Gy of orthovoltage radiation. The percentage of cells in each phase of the cell cycle was determined at various times after irradiation using flow cytometry. All cell lines underwent a dose-dependent arrest in G2 phase after irradiation, but the resistant cell lines underwent a significantly longer arrest in G2 phase after irradiation than did the sensitive cell lines. In conjunction with other results from our laboratories, we suggest that this difference in G2 arrest may be the basis for the increased resistance of cells transfected with oncogenes to irradiation.

Rat embryo cells (REC) transformed by the H-ras oncogene plus the cooperating oncogene v-myc are highly resistant to ionizing radiation as compared with the nontransformed parent cells, REC, or immortalized REC. In an attempt to understand the potential mechanism of resistance in these cells, the induction and repair of double strand breaks (dsb) in DNA were measured in a H-ras plus v-myc transformed (3.7) and an immortalized REC (mycREC) line using pulsed field gel electrophoresis. Cells were irradiated in the exponential phase of growth, and the amount of DNA dsb present was quantified by measuring the fraction of DNA activity released from the agarose plugs in which cells were embedded. Similar values of the fraction of DNA activity released were measured for both cell lines at equal X-ray doses, after correction for differences in cell cycle distribution, suggesting a similar induction of DNA dsb per Gy. Repair of DNA dsb measured after exposure to 40 Gy of X-rays was similar in both cell lines and displayed a fast and a slow component. The fast component had a 50% repair time of approximately 12 min, and the slow component, 50% repair time of about 3 h. These results suggest that the relative radioresistance of 3.7 cells is not conferred by a decrease in the amount of DNA dsb induced per Gy per dalton or by alterations in the capacity of the cells to repair DNA dsb. It is hypothesized that alterations in the expression of potentially lethal damage underlie this phenomenon.

The ras oncogene has been shown to induce metastatic potential in a wide variety of cells. However, one cell line, C127, when transformed by ras (HC127), proved to be an exception in that the transformed cells gained the ability to form tumors in nude mice, but these tumors did not form spontaneous metastases. Because C127 cells do not metastasize after ras transfection, these cells can be used to identify other factors which contribute to the development of metastatic potential. Specifically, these cells can be used as recipients in DNA transfer experiments utilizing DNA from other cells in which H-ras has been used to induce the metastatic phenotype, thus allowing the search for genes in addition to ras itself which may be necessary for metastasis. A system utilizing genomic DNA transfer into C127 cells transformed by ras has been developed. These cells were used as the recipient for genomic DNA in cotransfection with pSV2neo followed by selection both in the experimental i.v. assay and in the spontaneous metastasis assay in nude mice. DNA from two lines with metastatic potential (both transformed by ras genes) gave rise to metastatic clones, whereas DNA from the recipient cells, HC127, NIH 3T3 cells, and two human tumor cell lines, CHP126 and A2058, failed to give rise to transfectants which could metastasize. The metastases after the first cycle were put into culture, and DNA was extracted from these cells and used in a second cycle of transfection. The capacity to metastasize was also transferred in the second cycle. Of seven metastatic clones examined, four showed no detectable rearrangements of the transfected v-H-ras gene, but in three of these clones, there were rearrangements of this gene, which was originally present in the recipient cell, HC127. This indicates that in a subset of the selected clones there may have been selection for rearrangements of the host genome rather than introduction of foreign DNA sequences, and that such effects must be considered in gene transfer experiments. It is also possible that the transfer of particular genomic DNAs are leading to genetic instability in these experiments. mRNA levels were compared in the metastatic variants and the parental cells; H-ras-specific RNA was raised from 4- to 22-fold in the metastatic cell lines, regardless of rearrangement of the v-H-ras gene.

In a previous study we described a correlation between the metastatic potential of transformed rat embryo cells (REC) and their release of type IV collagenolytic activity (S. Garbisa et al., Cancer Res., 47: 1523-1528, 1987). In the present study, we have identified a Mr 92,000 gelatinase released exclusively by metastatic cell lines in vitro; seven of eight highly metastatic REC lines transformed by H-ras or H-ras plus v-myc released this enzyme. In contrast, the Mr 92,000 gelatinase was not detected in two separate nontumorigenic REC lines immortalized with H-ras or c-myc or in four independent tumorigenic REC lines (transformed by H-ras plus adenovirus E1A) with markedly reduced metastatic potential. Study of the Mr 92,000 gelatinase in tumor explants showed that its expression may be modulated in vivo. Not only was Mr 92,000 release enhanced in tumor explants from cell lines which released it in vitro, but its expression was evident in three primary tumors and six metastatic tumors from the one metastatic cell line that failed to release it in vitro. Explants from the nonmetastatic cell lines grown as tumors showed no Mr 92,000 gelatinase release. The heterogeneous expression of a number of other gelatinases with molecular weights of 52,000, 56,000, 62,000, 68,000, 80,000, and 240,000 was observed, but no correlation with metastatic potential was apparent. The Mr 92,000 gelatinase had the characteristics of a secreted neutral metalloproteinase. It may be responsible for the type IV collagenolytic activity reported previously in conditioned medium from metastatic transformed REC and could in part be responsible for the differences in metastatic potential in these cell lines.

The sensitivity of tumor cells to the killing effects of ionizing radiation is thought to be one of the major determinants of curability of tumors in patients treated with radiation therapy. This paper reviews the evidence from our laboratory and other groups which supports a role for oncogenes in the induction of radioresistance in cultured mammalian cells. Primary rat embryo cells (REC) were chosen as a model system in which the effects on radiation resistance of the H-ras oncogene could be studied on a uniform genetic background. These cells offered several useful advantages. The cells prior to transformation are diploid and because they have been in culture only for a few passages prior to transformation with the oncogene it is unlikely that any preexisting mutation affecting radiation response could be present. Additionally, the use of REC permitted the study of the effects of synergism between oncogenes on the induction of the radioresistant phenotype. The results show that the activated H-ras oncogene induces radiation resistance in primary rat cells after transformation, but that the effect of the oncogene itself is small. However, the myc oncogene, which has no effect on radiation resistance by itself, appears to have a synergistic effect on the induction of radiation resistance by H-ras. Radiation resistance induced by H-ras plus myc is characterized by an increase in the slope of the curve at high doses but there is also a large effect within the shoulder region of the radiation survival curve. The AdenoE1A oncogene which will also act synergistically with ras in transformation assays plays a less clear-cut role in assays of radiation resistance. The H-ras oncogene is also known not only to transform cells but also to induce metastatic behavior in the tumors which form after these transformed cells are injected into syngeneic animals or nude mice. We have also shown in our primary rat embryo cell system that the induction of metastatic behavior in transformed cells, like the induction of radioresistance depends on a complex interaction between oncogenes and the cellular background. This evidence will be reviewed to demonstrate some of the analogies between radiation resistance and metastasis as examples of the complex alterations in cellular phenotype which occur after oncogene transfection.(ABSTRACT TRUNCATED AT 400 WORDS)

Resistance of tumors to irradiation or chemotherapeutic agents is thought to be one of the reasons why patients who present with early malignancies may not be cured. Much is now known about the molecular mechanisms that underlie drug resistance, but until recently little was known about genetic contributions to radiation resistance. Some evidence now links oncogenes, particularly the ras family of oncogenes, to radiation resistance but heterogeneity between tumors and cell lines has complicated this analysis. Primary rat embryo cells have been chosen as a model system in which the effects on radiation resistance of the H-ras oncogene could be studied on a uniform genetic background. These cells offer several useful advantages. The cells prior to transformation are diploid, and because they have been in culture only for a few passages prior to transformation with the oncogene it is unlikely that any preexisting mutation affecting radiation response could be present. Additionally, the use of rat embryo cells permitted the study of the effects of a second oncogene on the appearance of the radioresistant phenotype. The results show that the activated H-ras oncogene is associated with radiation resistance in primary rat cells after transformation but that the effect of the oncogene by itself is small. However, the oncogene v-myc, which has no effect on radiation resistance by itself, has a synergistic effect on radiation resistance with H-ras. There appear to be differences in the phenotype of radiation resistance associated with these two forms of transfectants. Thus, radiation resistance seen with H-ras by itself is characterized by a change in the slope of the radiation survival curve at high radiation doses but little or no change within the should region of the radiation survival curve. Radiation resistance seen in H-ras plus v-myc transformants is also characterized by an increase in the slope of the curve at high doses but there is also a large effect within the shoulder region of the radiation survival curve. These studies led to the following conclusions: (a) the radioresistant phenotype is not due to preexisting genetic heterogeneity in the cells prior to transfection; (b) the radiation resistant phenotype of cells transformed by H-ras is seen to a greater degree in cells which also contain the v-myc oncogene; (c) the v-myc oncogene may play an important role in the phenotype of radiation resistance at low doses that is within the range most critical for clinical practice.

Second-passage rat embryo cells were transfected with a neomycin resistance gene and the activated form of the c-Ha-ras I gene, or with these two genes plus the adenovirus type 2 E1a gene. Foci of morphologically transformed cells were observed in both cases; however, the frequency of transformation was at least ten times higher with two oncogenes than with the ras gene alone. All the transformed cell lines gave rise to rapidly growing tumors when injected subcutaneously into nude mice. All but one of the cell lines transformed by the ras oncogene alone formed metastatic nodules in the lungs of animals that had been injected subcutaneously with transformed cells. When transformed cells were injected intravenously, all the ras single-gene transformants gave rise to many metastatic lung nodules. In contrast, cell lines transformed with ras and E1a did not generate metastases after subcutaneous injection and gave rise to very few metastatic lung nodules after intravenous injection. These data demonstrate that a fully malignant cell with metastatic potential, as measured in an immunodeficient animal, can be obtained from early passage embryo cells by the transfection of the ras oncogene alone.

Cancer invasion and metastases is a complex multi-step process. In order for a tumor cell to successfully traverse all the steps of this process and initiate a metastatic colony, it must express the right combination of gene products. Such gene products may include proteins which regulate cell interaction with the basement membrane and cell motility. Tumor cells attach to the basement membrane glycoprotein laminin via the cell surface laminin receptor. The human laminin receptor was purified and molecularly cloned. The level of laminin receptor mRNA in a variety of human carcinoma cells correlated with the number of laminin receptors on the surface of these cells. Following attachment to the basement membrane, the tumor cell next secretes proteases which may degrade type IV collagen. A genetic linkage between type IV collagenase secretion and metastases was collagen. A genetic linkage between type IV collagenase secretion and metastases was studied using our new genetic system for inducing metastases by employing the ras oncogene. Following attachment and local proteolysis, the third step of invasion is tumor cell motility. We have isolated a tumor cell autocrine motility factor (AMF). This factor is secreted by the tumor cells and binds to a cell surface receptor, resulting in a profound (greater than 100 x) stimulation of cell locomotion. AMF may play a major role in the autonomous invasive behavior of tumor cells.

The hypothesis of tumor progression proposed by Nowell [P. C. Nowell, Science (Wash. DC), 194: 23-28, 1976] states that one mechanism for the development of the metastatic phenotype could be the induction of chromosomal instability. We have developed a new experimental system for studying the induction of the metastatic phenotype using early passage fibroblasts which become metastatic in nude mice after transformation with the Harvey ras oncogene [R. J. Muschel et al., Am. J. Pathol., 121: 1-8, 1985; R. Pozzatti et al., Science (Wash. DC), 232: 223-227, 1986]. Since the early passage fibroblasts themselves are diploid, we reasoned that this might be a system in which karyotypic change after tumor formation or metastasis might easily be evaluated. Thus, we performed cytogenetic analysis on multiple metastases and tumors which had been derived from cells transformed with the cellular Harvey ras1 oncogene and compared their karyotypes. The karyotypes of the cells isolated from 5 tumors and 14 metastases were, as far as we could determine, identical to those of the injected cells. This could easily be evaluated because of the two clones studied one was diploid; the other has a trisomy of chromosome 4 without any other detectable abnormality. These results suggest that in this system using nude mice, selection for a necessary or even advantageous chromosomal aberration does not occur during tumor formation or metastasis. Furthermore, they indicate that the presence of the ras gene itself does not induce chromosomal rearrangements or aneuploidy and that a cell can be both tumorigenic and metastatic yet remain diploid.

NIH-3T3 cells and early passage fibroblasts transformed by various members of the rasH gene family were found to express metastatic potential in nude (Nu/Nu) mice. NIH-3T3 cells transformed by either the cloned DNA of the Harvey sarcoma virus or by the T24 human rasH oncogene were both tumorigenic after subcutaneous injection and metastatic after intravenous injection. In contrast, NIH-3T3 cells transformed by elevated levels of the proto-oncogene were tumorigenic but not metastatic. Tumor growth potential by itself, therefore, was not sufficient to induce metastatic behavior. Diploid fibroblasts transformed with the T24 rasH oncogene were tumorigenic and metastatic after intravenous injection and also produced extensive spontaneous metastases. Thus, induction of the complete metastatic phenotype by the T24 rasH oncogene does not require the use of aneuploid recipient cells such as NIH-3T3 cells. An alternative murine recipient cell type C127, when transformed by the cloned DNA of the Harvey sarcoma virus, was tumorigenic but not metastatic. The transformed C127 cells made less of the viral gene product P21 than the NIH-3T3 rasH transformants. To ensure that the decreased levels of P21 were not responsible for the lack of metastatic potential, C127 cells were transformed with altered constructs of the plasmid containing the Harvey sarcoma virus which elicited enhanced levels of P21. The augmented P21 levels, although equivalent to or greater than that seen in the NIH-3T3 transformants, did not confer metastatic potential on the C127 transformants. These results indicate that at least two complementation groups may be required for induction of metastatic capacity in this system, one involving the "activated" oncogenic form of the rasH gene and the second another as yet undefined factor in the cellular background present in NIH-3T3 cells but absent in C127 cells.

A karyotypic analysis was performed on seven independently derived clones of primary rat embryo cells transformed by the ras oncogene plus the cooperating oncogene myc. The transfected oncogenes were sometimes present in amplified copy number, with heterogeneity in the levels of amplification. Some chromosomal features, such as aberrantly banding regions and double-minute chromosomes, typical of cells carrying amplified genes, were also seen in three of the seven cell lines. Underlying this heterogeneity there was an unexpected finding. All seven lines showed a common integration site for ras on the q arm of rat chromosome 3 (3q12), though some lines also had other sites of integration. In four of the lines integration of ras was accompanied by deletion of the p arm of chromosome 3 or its possible translocation to chromosome 12.

Studies on genetically selected mutants in phagocytosis of E[IgG] indicated that the defect in some mutants could be corrected by addition of 8 Br-cAMP, and suggested that cyclic AMP may be involved in the mechanism of phagocytosis through the Fc receptor. In order to elucidate the role of cyclic AMP in phagocytosis in the parental, nonmutant macrophage-like cell line, J774.2, it was necessary to employ restrictive conditions which rendered phagocytosis suboptimal. When 4774.2 cells were cultured in nontissue culture Petri dishes phagocytosis was markedly reduced. Addition of 8 Br-cAMP or inducers of intracellular cyclic AMP such as isoproterenol restored the phagocytic ability of these cells. Similarly, treatment of the parental J774.2 cells with insulin reduced the level of phagocytosis, and once again this suppression could be corrected by addition of 8 Br-cAMP. In no case did AMP mimic the effects of 8 Br-cAMP. The effect of cyclic AMP action in this system was not instantaneous, but rather reached optimal levels at 5 to 10 hr, suggesting that cyclic AMP is not the immediate signal for phagocytosis. The genetic analysis of macrophage variants may provide a useful model for studies on the mechanisms of phagocytosis, and also the effects of insulin and cyclic AMP on an easily measurable biologic function in a specialized cell type.

Although tumors are often characterized as being the result of uncontrolled proliferation, clinically the morbidity from cancer results from its ability to invade and metastasize and to become resistance to our current therapeutic modalities such as chemotherapy and radiation. The study of oncogenes has largely stressed their ability to alter cell growth, but in fact they also influence these crucial features of tumor progression. In this review, we will summarize the data from our laboratory that indicated that the ras oncogene can induce metastasis as well as tumorigenicity and that the ras oncogene can also influence the development of radiation resistance. We have found that these changes in cells are sometimes accompanied by characteristic non-random chromosomal alterations in the transformed cells and will speculate that these accompanying chromosomal changes also may contribute to the features of tumor progression.