ABSTRACT

Recent studies have implicated a number of oncogenes such as int , c-erb B2, c- myc , and ras in the development of mouse, rat and human mammary tumors. The present study investigated whether or not the newly discovered jun oncogene is also involved in such tumors. In order to achieve this goal, RNA from mammary tumors and normal mammary tissues was isolated, electrophoresed in agarose gels, and hybridized with mouse mammary tumor virus (MMTV) and jun oncogene specific probes. The results show that, in general, jun B expression is higher in mammary tumors and in lactating mammary glands as compared to normal non-lactating mammary tissues of laboratory mice. Expression of jun B is also high in the mammary tumors of MMTV- infected JYG mice derived from the wild, and in chemical carcinogen-induced mammary tumors of rats. Furthermore, jun B RNA was found to be present at high levels in human breast tumors. Interestingly, 4 of the 5 human breast tumors tested showed, in addition to the 2.1 kb jun transcript, two other 1.8 and 1.5 kb transcripts which have not been previously reported. Therefore, although jun B is ubiquitously expressed in mammary tissues, its presence in higher amounts in mammary tumors and lactating mammary glands suggests that it may play a role in mammary cell proliferation and/or in mammary tumorigenesis.

INTRODUCTION

The discovery that normal cells contain genes which have the ability to induce tumors marks a turning point in cancer research (1). These cancer causing genes are called protooncogenes or oncogenes. To date, more than 30 oncogenes have been discovered, and it is believed that there are more oncogenes yet to be identified. Although most of the presently known oncogenes have been found to be associated with a variety of animal tumors, formal demonstration of the involvement of many of these genes in tumorigenesis is lacking. However, numerous results now suggest that alterations in either the structure, copy number, or expression of these genes lead to the transformation of a variety of normal cells including mammary cells to malignant tumor cells (2).

The development of spontaneous mammary tumors in mice is frequently associated with chronic infection of host mammary tissue by a retrovirus called mouse mammary tumor virus (MMTV) which induces mammary tumors by acting as an insertional mutagen within the mammary epthelial cells. The expression of at least three cellular genes, int -1 (also designated by Wnt- 1; 3), int - 2 (Fgf- 3; 4), and int -3 (5), which are not normally expressed in the mammary gland, are activated in mammary tumors as a consequence of the integration of an MMTV proviral genome into flanking cellular DNA sequences (5-8). One of the oncogenes, int -2 has been implicated in human breast cancer because it is amplified in breast tumor tissues but not in normal tissues (9). In addition, a number of other oncogenes, such as erb B2 (also known as c-neu or HER -2), and p53, have also been implicated in human breast carcinogenesis (10-16). It is of interest to note that many oncogenes are believed to act singly or in combination with other oncogenes in inducing different types of tumors in different hosts (17). For an example, erb B2 was initially isolated from rat neuroglioblastomas (18), but now it has been implicated in both human breast cancer and ovarian cancer (19,20). Similarly the ras oncogene that was initially cloned from a human bladder carcinoma (21) has now been shown to be associated with a variety of tumors including mammary tumors in rats (22,23). It is possible, therefore, that human breast carcinogenesis may result from the activaton of a number of the presently known oncogenes, including the BCR I gene (for review see 24), as well as other oncogenes yet to be identified.

The oncogene jun has presently become one of the best-known oncogenes because of its ability to act as a transcription factor (25,26). It has been shown that jun comprises a set of related genes, c-jun or jun C, jun B and jun D (27-29). Regarding the expression of jun, Hirai et al. (28) examined the mRNA levels of jun C, jun B and jun D in various mouse tissues and concluded that each of these genes is expressed independently in different tissues and that they may play a role in growth, development and cellular differentiation. Unfortunately, this study did not include mammary tissues, especially mammary tumors. The present study was therefore undertaken to examine the levels of jun expression, especially jun B, in normal and tumorous mammary tissues of mice and rats, and in human mammary tumors.

MATERIALS AND METHODS

Tissues:
MMTV-infected and high mammary tumor-producing C3H/HeJ and C3H/OuJ mice were purchased from Jackson Laboratory, Bar Harbor, Maine. Mammary tumors of JYG mice were obtained from Dr. S. Imai of the Nara Medical University, Japan (8,30). Normal mammary glands and mammary tumors from a number of mouse strains, BALB/c, BALB/cfC3H, C57BL, C57BLfGR, GR and RIII/Sa, were collected from mice bred in our own laboratory. BALB/c and C57BL mice do not carry any exogenous (MMTV) and do not develop spontaneous mammary tumors. RIII/Sa and GR mice are MMTV-positive and have a 90-100% tumor incidence. RIII/Sa is a subline of RIII/IMR mice (31). BALB/cfC3H and C57BfGR mice were developed by foster nursing BALB/c and C57BL neonates on C3H and GR mice, respectively. These mice express high levels of MMTV and have a high incidence of mammary tumors. Mammary tissues from WLCO mice were the gift of Dr. D. Morris, University of California, Davis, California. These mice, derived from a pair of wild mice, neither carry MMTV nor develop tumors (Dr. Morris, personal communication). 7,12 dimethylbenz(a)anthracene (DMBA) induced rat tumors (32) and normal rat mammary glands were obtained from Dr. D. Kritchevsky, Wistar Institute, Philadelphia, PA. Human breast tumors were provided by Dr. P. Etkind, Albert Einstein College of Medicine, Bronx, New York.

Cellular RNA extraction:
Total cellular RNA was isolated from frozen mammary tissues (0.5 to 0.9 g) by using the lithium chloride/urea method (33). Common chemicals in this study were obtained from Sigma (St. Louis, MO). The tissues were placed in 10 volumes of ice cold 3M LiCl/6M urea and homogenized with a Brinkmann polytron homogenizer at 27,000 rpm for approximately 1 min. The homogenate was placed in an ice water bath overnight. The extract was centrifuged at 10,000 g at 4 ° C for 1 hr and the pellet was suspended in 10 mM Tris (pH 7.5), 10 mM EDTA, 0.5% SDS, and 200 µg/ml proteinase K at room temperature for 15 min during which time the material became evenly suspended. One-tenth volume of 2.5 M NaOAc (pH 5.5) was added followed by equal volumes of phenol-chloroform. The solution was gently shaken for 10 min and centrifuged for 10 min in a tabletop centrifuge at 3000 rpm. The aqueous phase was removed and re-extracted with phenol- chloroform twice, followed by one extraction with chloroform only. The solution was stored at -20 ° C in an equal volume of isopropanol. Aliquots were spun down and the concentration and purity of the RNA sample was determined using a Shimadzu spectrophotometer.

Preparation of DNA probes for hybridization:
Recombinant plasmids containing the sequences for MMTV, jun B and jun C, and chicken-actin were used for the preparation of DNA probes. These respective plasmids were obtained from the laboratory of H. Varmus of the University of California, San Francisco (6), the American Type Culture Collection, Rockville, Maryland, and M. Sudol of the Rockefeller University, New York (34). The plasmid DNAs that contained the MMTV (9.8 kb), jun C (2.8 and 2.6 kb) and jun B (2.9 kb) inserts were digested with the restriction enzyme Eco RI, while the plasmid DNA that contained the actin insert was digested with Pst I. The size of the actin probe was 300bp. The digests were electrophoresed on a 0.8% agarose gel, the inserts were visualized under UV light and the portion of the gel containing the inserts were cut out, and the insert DNAs were electroeluted. The DNAs were then concentrated using DEAE cellulose columns (Elutip-d, Schleicher and Schuell, Keene, NH), precipitated with two volumes of ethanol at -20 ° C overnight, centrifuged, washed once with 70% ethanol and the DNA pellet dried with a stream of N2, and finally resuspended in a desired volume of HEPES buffer, pH 7.5. The purified inserts were nick translated using 32P-deoxynucleotides as labeling substrate following the procedures described by Sambrook, et al (35). Briefly, 10 µl of 32P-CTP (New England Nuclear, Boston, MA, sp. act. 300-500 Ci/mmole) were dried under vacuum. At the same time a "mix" was prepared which contained 50 mM KPO4, pH 7.0 mM MgCl2, 10 mM dGTP, 10 mM dTTP, 10 mM dATP, 50 µg/ml BSA (Pharmacia, Piscataway, NJ) 10 mM 2-mercaptoethanol, and 10 ng/ml DNase I (Sigma). To the dried nucleotides was added 20 µl "mix", 0.1 µg of DNA insert and 0.2 µl DNA polymerase I (5 units, Sigma). The reaction mixture was incubated for 60 min at 15 ° C. The nick translated DNA was then loaded onto a Sephadex G-50 column in a buffer containing 0.15 M NaCl, 10 mM Tris, pH 7.9, 1 mM EDTA, and 0.1% SDS, which was then centrifuged at 1,000 x g for 5 min at 10 ° C, and the labeled DNA solution drawn into the centrifuge tube was collected. The specific activity of the probe was ascertained by liquid scintillation counting.

Agarose Gel Electrophoresis of RNA:
For analysis of RNA, agarose gels (0.8%) containing 2.2 M formaldehyde and 10 x MOPS buffer were prepared as described (35). In brief, RNA samples of 25 µg were centrifuged and washed with 70% ethanol. The precipitate was dissolved in 6 µl of DEPC- treated water and 21 µl of RNA sample buffer (contains 100 µ l formamide, 35 µl formaldehyde and 20 µl of 10 x MOPS buffer). The samples were denatured in a 65 ° C hot water bath for 10 min and briefly cooled on ice. A 3 µl solution containing 20% Ficoll, 10 mM EDTA (pH 8.0), 0.25% zylene cyanol and 0.25% bromophenol blue was added to the RNA preparation and loaded into the gel. The gel was run for approximately 5-6 hrs. at 100 V with continuous circulation of buffer.

Transfer of RNA to Zetaprobe Nylon Filters:
Post electrophoresis, the gel was rinsed in DepC treated water for 30 minutes followed by transfer to a Zeta Probe membrane as described (35). Post transfer, the filters were washed for 5 min in 2 x SSC and 0.1% SDS with agitation, and the RNA cross-linked at an energy of 1200 f Joules/100 sq cm area with a Stratagene UV Stratalinker 2300.

Prehybridization and Hybridization of RNA:
Also as previously described (35), non-specific binding was then blocked by membrane placement into 20 ml of prehybridization solution ( 0.2 M NaH2PO4 (pH 7.2), 1 mM EDTA, 1% BSA, 7% SDS, and 45% formamide.) into heat-sealable bags at 65 ° C with agitation for a minimum of 2 hrs. A minimum of 1 x 107 cpm of the appropriate oligolabelled DNA probe was denatured, rapidly chilled, and added to the bag containining the prehybridized membrane. Hybridization was then carried out by incubation for 18-24 hrs. at 65 ° C. The membrane was then removed from the bag and washed three times in 500 ml of 40 mM NaH2PO4, 1 mM EDTA, 1% SDS for a total of 2 hours. The membrane was then placed in a fresh heat-sealable bag and exposed to Kodak XAR-5 film at -70 ° C with a Dupont intensifying screen. After autoradiography, the radioactive probe was removed (dehybridized) from the filter by boiling which enabled the filter to be rehybridized with a second probe.

RESULTS

Expression of MMTV and jun RNA.

a) Normal Mammary Glands and Mammary Tumors of Mice and Rats:
It is known that mammary tumors in mice are caused by a retrovirus, the mouse mammary tumor virus (MMTV). It is also known that the level of virus expression increases with age and parity of the mouse (36). The goal of the present work was to determine if the jun oncogene is expressed in mammary glands and if so, to evaluate whether its expression changed with the development of mammary tumors. To accomplish this objective, RNA was extracted from various tissues, and analyzed by Northern blotting. (Only samples which were found to have intact 28S and 18S ribosomal bands as judged by staining with ethidium bromide were used for hybridization). Samples judged suitable for Northern analysis were first hybridized with an MMTV rep probe (6) (Fig. 1, Panel A), followed by stripping and rehybridization with a jun C probe (Fig. 1, Panel B), followed by a final stripping and rehybridization with an actin probe (Fig. 1, Panel C).

As shown in Figure 1, both mammary tumors (Panel A, lanes 1 and 2) and normal mammary glands from mice infected with MMTV (RIII/Sa and BALB/cfC3H) express, as expected, two MMTV transcripts, one of which represents the genomic RNA (8.6 kb) while the other (3.8 kb) represents the mRNA for the viral envelope proteins (31). The tissue samples also expressed two c-jun specific 3.2 and 2.7 kb transcripts. The level of expression of both MMTV and c-jun seemed to be higher in mammary tumors and lactating mammary glands (lanes 1-3) than that in a non-lactating mammary gland, since the relative amount of mRNA from each tissue sample loaded into the gel appears to be similar. This conclusion is based on the observation that the actin probe hybridized to all four RNA samples with similar intensity (Panel C).

Having established that both mammary tumors and normal mammary tissue expresses jun C, an attempt was made to determine if they also express jun B, a closely related member of the jun family. As shown in figures 2 and 3, indeed both mammary tumors and normal mammary glands of mice express high levels of jun B (also see Table I). Interestingly, however, lactating mammary glands of mice seem to express a higher level of jun B compared to non-lactating mammary glands. This observation is similar to what was observed for jun C. Additionally, the level of jun B RNA in mammary tussues of MMTV-negative mouse strains, BALB/c and C57BL, is low, but following MMTV infection the mammary glands of these mice (BALB/cfC3H and C57BLfGR) appeared to express more of the jun B transcripts.

In contrast to the above observations, the normal mammary glands of WLCO mice, derived by breeding from a pair of wild mice, which neither carry endogenous/exogenous MMTV nor develop mammary tumors were found to express the lowest amount of jun B RNA (at least 20-30 fold less than that expressed by several conventional laboratory strains of mice). On the other hand, mammary tumors of another wild-mouse-derived strain of mice (JYG), infected with MMTV (D. Morris, personal communication) expressed jun B in abundance (Fig. 4). Studies of the expression of jun B in rat tissue showed that both mammary tumors and mammary glands contained jun B RNA. The levels of RNA, however, were found to be slightly higher in mammary tumors (1.2-1.7x) than in normal tissue.

b) Human Mammary Tumors:
There is evidence for the presence of MMTV related sequences in human DNA (37-40). Further, human milk and human breast cancer cells have been found to contain retrovirus-like particles and antigens which are recognized by antibodies prepared against the major glycoprotein of MMTV (41-43). However, the human breast tumors tested in the present study were not found to express any MMTV transcript (Fig. 5, Panel B).

In view of the notion that a number of oncogenes, are involved in human mammary tumorigenesis, we determined whether or not human breast tumors express jun .. Total cellular RNA from human breast tumors were isolated, size fractionated by agarose gel electrophoresis, transferred to filters and hybridized with jun B probe. Our results show that all of the five tumors tested expressed the 2.1 kb jun B (Fig. 5, Panel A) transcript. In addition, the jun B probe, used in the present study, detected two other species of RNA, 1.8 kb and 1.5 kb in sizes, in 4 of the 5 human tumors. This observation is unique in that none of the several normal or cancerous tissues from mouse and rats that we analyzed showed the presence of the 1.8 kb and 1.5 kb jun B specific transcripts. It should be noted that no previous studies have reported these transcripts in any other tissues.

The Levels of the Expression of MMTV and jun B RNA in Different Tissues:

In order to compare the relative levels of the expression of MMTV and/or jun B RNA in different tissue samples, each filter was sequentially hybridized with MMTV, jun B, and actin probes and the intensities of hybridization of the MMTV and jun B transcripts were compared with the intensities of hybridization of the corresponding samples with the actin probe. Our qualitative and quantitative evaluations of the autoradiograms, using actin RNA as a standard, revealed that the levels of the expression of MMTV and jun B RNAs varied from one sample to another. Some examples of our analyses are illustrated in Fig. 6. As judged visually, the intensity of the actin band in lane 12 is about 10-fold higher than that of the intensity of the jun B band in the corresponding lane 4. By contrast, the intensity of hybridization produced by the jun B RNA in lane 5 is more than 3-fold the intensity of hybridization produced by the actin RNA in lane 13. It should be noted also that the intensity of the actin band in lane 12 is similar to that in lane 13 and thus the amount of the RNA samples loaded in lanes 4 and 5 were nearly equal. From these observations, it is quite clear that the amount of jun B RNA present in lane 5 was several-fold higher than was present in the same amount of total cellular RNA loaded in lane 4. We present another example of how we ascertained the relative levels of the expression of MMTV or jun B RNA in those samples that produced very similar signals of hybridization, such as the samples numbered 6 and 7. The density of the jun B bands (areas A and B) representing the sample number 6 (lane 6) and 7 (lane 7), and the actin bands (areas C in lane 14, and D in lane 15) corresponding to the same samples were determined. Using these data it was estimated (area A x area D / area B x area C) that the sample number 6 contained 1.6-fold more jun B transcripts than the sample number 7. The results of our evaluation of the relative levels of the expression of MMTV and jun B transcripts in various breast tumors and normal mammary glands that we examined are shown in Table 1. For comparative purposes we have used the symbols 1+, 2+, 3+ and 4+ to represent constitutive, 1.5-2.5, 2.6-5.0, and more than 5-fold higher than the constitutive levels of expression. Our results clearly show that MMTV and jun B expression varies significantly among mice, rats, and humans.

DISCUSSION

The results presented in this report show that mammary tissues from mouse, rat, and human contain constitutive levels of jun B transcripts. This is not surprising since in a previous survey that did not include mammary tissue, jun B was found to be present in a variety of mouse tissues (28, 44, 45). What is interesting is that lactating mammary glands and mammary tumors of certain strains of mice, and the mammary tumors of rats and humans were found to contain higher levels of jun B. In addition, our results also show correspondence between the levels of the expression of jun B and MMTV. Analyses of the human breast tumors also revealed an interesting finding: 4 of the 5 tumors tested contained two additional 1.5 and 1.8 kb jun B transcripts in addition to the known 2.1 transcript.

The jun oncogene was first isolated from a spontaneous sarcoma in an adult chicken (46). The known members of the jun family code for proteins which regulate gene expression directly by binding to a common cellular and viral DNA sequence motif, TGACTCA, or indirectly via dimerization and cooperativity with the product of another oncogene, fos . Although viral jun (v- jun ) is an efficient transforming agent in chickens, little work has been done demonstrating the ability of either v-jun or c-jun to transform in mammalian cells. However, one study has shown that primary cultures of embryonic rat cells became morphologically transformed and acquired tumorigenic potential when they are co-transfected with a leukemia retroviral vector carrying a human c-jun gene and an activated ras gene (47). In another study, the jun gene alone was shown to transform established rat fibroblastic cell lines (48). Based on these observations, it has been speculated that overexpression of jun in normal mammalian cells may complement other constitutive growth signals to bring about oncogenic transformation even though jun itself may not be sufficient for oncogenesis in mammalian system (25).

Since jun is known to be activated by external growth signals, it is likely that jun, in conjunction with fos and probabaly other proteins, may alter transcription of specific genes, thereby triggering the cellular growth response. These speculations are in accord with the present observations that lactating mammary glands and mammary tumors show elevated levels of jun expression. In the case of lactating mice, the mammary cells are obviously under the influence of hormones which may provide growth signals triggering alterations in the transcriptions of a variety of genes including jun .

At present, the significance of higher levels of jun expression in mammary tumor cells remains unknown. However, in view of the finding that small lung cell carcinomas of humans contain higher concentrations of jun transcripts, one may speculate that a similar situation may exist in other types of cancer. The present study thus provides a basis for further investigations into the role of enhanced expression of jun gene in mammary cells. Although the results presented in this report show a definitive trend of increased jun expression in lactating mammary glands and mammary tumors as compared to non-lactating mammary tissues, more quantitative data are needed to establish a definitive relationship between the levels of jun expression and mammary cell transformation. Furthermore, the two new jun B transcripts, 1.5 and 1.8 kb, that were found to be present only in human breast tumors raises interesting questions about their synthesis and function.

CONCLUSIONS

In conclusion, although the transcription factor jun B is found to be indiscriminately expressed in the mammary tissues of mice, rats, and humans, its increased presence in mammary tumors and lactating mammary glands indicate that jun B may play an important role in mammary cell differentiation and transformation.

ACKNOWLEDGEMENTS

Thanks are due to Dr. A. Hossain of the Medical College of Georgia for his help and advice. This work was partly supported by grants from the National Institutes of Health (CA- 45127), American Institute for Cancer Research (92B31), and the American Cancer Society (BC- 494).

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