Immortalization of hypothalamic GnRH neurons by genetically targeted tumorigenesis.

By genetically targeting tumorigenesis to specific hypothalamic neurons in transgenic mice using the promoter region of the gonadotropin-releasing hormone (GnRH) gene to express the SV40 T-antigen oncogene, we have produced neuronal tumors and developed clonal, differentiated, neurosecretory cell lines. These cells extend neurites, express the endogenous mouse GnRH mRNA, release GnRH in response to depolarization, have regulatable fast Na+ channels found in neurons, and express neuronal, but not glial, cell markers. These immortalized cells will provide an invaluable model system for study of hypothalamic neurosecretory neurons that regulate reproduction. Significantly, their derivation demonstrates the feasibility of immortalizing differentiated neurons by targeting tumorigenesis in transgenic mice to specific neurons of the CNS.

Immortalization of osteoclast precursors by targeting Bcl -XL and Simian virus 40 large T antigen to the osteoclast lineage in transgenic mice.

Cellular and molecular characterization of osteoclasts (OCL) has been extremely difficult since OCL are rare cells, and are difficult to isolate in large numbers. We used the tartrate-resistant acid phosphatase promoter to target the bcl-XL and/or Simian Virus 40 large T antigen (Tag) genes to cells in the OCL lineage in transgenic mice as a means of immortalizing OCL precursors. Immunocytochemical studies confirmed that we had targeted Bcl-XL and/or Tag to OCL, and transformed and mitotic OCL were readily apparent in bones from both Tag and bcl-XL/Tag mice. OCL formation in primary bone marrow cultures from bcl-XL, Tag, or bcl-XL/Tag mice was twofold greater compared with that of nontransgenic littermates. Bone marrow cells from bcl-XL/Tag mice, but not from singly transgenic bcl-XL or Tag mice, have survived in continuous culture for more than a year. These cells form high numbers of bone-resorbing OCL when cultured using standard conditions for inducing OCL formation, with approximately 50% of the mononuclear cells incorporated into OCL. The OCL that form express calcitonin receptors and contract in response to calcitonin. Studies examining the proliferative capacity and the resistance of OCL precursors from these transgenic mice to apoptosis demonstrated that the increased numbers of OCL precursors in marrow from bcl-XL/Tag mice was due to their increased survival rather than an increased proliferative capacity compared with Tag, bcl-XL, or normal mice. Histomorphometric studies of bones from bcl-XL/Tag mice also confirmed that there were increased numbers of OCL precursors (TRAP + mononuclear cells) present in vivo. These data demonstrate that by targeting both bcl-XL and Tag to cells in the OCL lineage, we have immortalized OCL precursors that form bone-resorbing OCL with an efficiency that is 300-500 times greater than that of normal marrow.

Two distant and precisely positioned domains promote transcription of Xenopus laevis rRNA genes: analysis with linker-scanning mutants.

To examine the internal organization of the promoter of the Xenopus laevis rRNA gene, we constructed a series of linker-scanning mutants that traverse the rDNA initiation region. The mutant genes, which have 3 to 11 clustered base substitutions set within an otherwise unaltered rDNA promoter sequence, were injected into Xenopus oocyte nuclei, and their transcriptional capacity was assessed by S1 nuclease analysis of the resultant RNA. The data demonstrate that there are two essential promoter domains, the distal boundaries of which coincide with the promoter boundaries established previously by analysis of 5' and 3' deletion mutants. The upstream promoter domain is relatively small and extends from residues ca. -140 to -128. The downstream domain is considerably larger, encompassing residues ca. -36 to +10, and exactly corresponds in both size and position to the mammalian minimal promoter region. The Xenopus rDNA sequence between these two essential domains has a much smaller effect on the level of transcriptional initiation. In light of the fact that a large portion of this intervening region consists of a segment (residues -114 to -72) that is duplicated many times in the upstream spacer to form an rDNA enhancer sequence, it is noteworthy that a "-115/-77 linker scanner," in which virtually this entire segment is replaced by a polylinker sequence, has full promoter activity in the injected Xenopus borealis oocytes. Analysis of a parallel series of spacing change linker-scanning mutants revealed the unexpected result that the relative positions of the upstream and downstream promoter domains are very critical: all spacing alterations of more than 2 base pairs within this 100-base-pair region virtually abolish promoter activity. We conclude that the factors that bind to these two distant promoter domains must interact in a very precise stereospecific manner.

The Xenopus ribosomal DNA 60- and 81-base-pair repeats are position-dependent enhancers that function at the establishment of the preinitiation complex: analysis in vivo and in an enhancer-responsive in vitro system.

Although it is generally believed that the 60- and 81-base-pair (60/81-bp) repeats of the Xenopus laevis ribosomal DNA (rDNA) spacer are position-independent transcriptional enhancers, this has not been shown directly. We have now developed a critical assay which proves that the 60/81-bp repeats do, in fact, stimulate transcription from promoters in cis and that they function in both orientations and when up to 1 kilobase pair from the initiation site. However, contrary to the widely accepted view, these elements are found to be highly position dependent, for they have no net effect when downstream of the initiation site within the transcribed region and they behave as transcriptional silencers of promoters in cis when moved greater than 2 kilobase pairs upstream of the initiation site. The 60/81-bp elements therefore are position-dependent 5' enhancers. We also found that this rDNA enhancer was polymerase I specific and that it was composed of duplicated, individually functional elements. Finally, we report an in vitro system that reproduces both cis enhancement and trans competition by the 60/81-bp repeats. Sequential-addition studies in this system demonstrated that the rDNA enhancer functions in trans at or before establishment of the stable transcription complex, not subsequently at each round of transcription.

Tissue-specific gene expression in the pituitary: the glycoprotein hormone alpha-subunit gene is regulated by a gonadotrope-specific protein.

The molecular mechanisms for the development of multiple distinct endocrine cell types in the anterior pituitary have been an area of intensive investigation. Though the homeodomain protein Pit-1/GHF-1 is known to be involved in differentiation of the somatotrope and lactotrope lineages, which produce growth hormone and prolactin, respectively, little is known of the transcriptional regulators important for the gonadotrope cell lineage, which produces the glycoprotein hormones luteinizing hormone and follicle-stimulating hormone. Using transgenic mice and transfection into a novel gonadotrope lineage cell line, we have identified a regulatory element that confers gonadotrope-specific expression to the glycoprotein hormone alpha-subunit gene. A tissue-specific factor that binds to this element is purified and characterized as a 54-kDa protein which is present uniquely in cells of the gonadotrope lineage and is not Pit-1/GHF-1. The human and equine alpha-subunit genes are also expressed in placental cells. However, the previously characterized placental transcription factors designated TSEB and alpha-ACT are not found in the pituitary gonadotrope cells, indicating that independent mechanisms confer expression of these genes in the two different tissues.

Increased tumor proliferation and genomic instability without decreased apoptosis in MMTV-ras mice deficient in p53.

We have used an in vivo tumor model to evaluate the consequences of p53 tumor suppressor protein deficiency in a tissue-specific context. By breeding MMTV-ras transgenic mice, which are highly susceptible to the development of mammary and salivary tumors, with p53(-/-) mice, we generated three classes of animals which contained the MMTV-ras transgene but differed in their p53 functional status (ras/p53(+/+), ras/p53(+/-), or ras/p53(-/-)). ras/p53(-/-) mice developed tumors more rapidly than animals of the other two genotypes; however, the distribution of tumors was unexpectedly altered. Whereas the most frequently observed tumors in ras/p53(+/+) and ras/p53(+/-) mice were of mammary origin, ras/p53(-/-) mice developed primarily salivary tumors. In addition, the mammary and salivary tumors from ras/p53(-/-) mice consistently exhibited a number of unfavorable characteristics, including higher histologic grades, increased growth rates, and extensive genomic instability and heterogeneity, relative to tumors from ras/p53(+/+) mice. Interestingly, the increased growth rates of ras/p53(-/-) tumors appear to be due to impaired cell cycle regulation rather than decreased apoptosis, suggesting that p53-mediated tumor suppression can occur independent of its role in apoptosis.

A farnesyltransferase inhibitor induces tumor regression in transgenic mice harboring multiple oncogenic mutations by mediating alterations in both cell cycle control and apoptosis.

The farnesyltransferase inhibitor L-744,832 selectively blocks the transformed phenotype of cultured cells expressing a mutated H-ras gene and induces dramatic regression of mammary and salivary carcinomas in mouse mammary tumor virus (MMTV)-v-Ha-ras transgenic mice. To better understand how the farnesyltransferase inhibitors might be used in the treatment of human tumors, we have further explored the mechanisms by which L-744,832 induces tumor regression in a variety of transgenic mouse tumor models. We assessed whether L-744,832 induces apoptosis or alterations in cell cycle distribution and found that the tumor regression in MMTV-v-Ha-ras mice could be attributed entirely to elevation of apoptosis levels. In contrast, treatment with doxorubicin, which induces apoptosis in many tumor types, had a minimal effect on apoptosis in these tumors and resulted in a less dramatic tumor response. To determine whether functional p53 is required for L-744,832-induced apoptosis and the resultant tumor regression, MMTV-v-Ha-ras mice were interbred with p53(-/-) mice. Tumors in ras/p53(-/-) mice treated with L-744,832 regressed as efficiently as MMTV-v-Ha-ras tumors, although this response was found to be mediated by both the induction of apoptosis and an increase in G1 with a corresponding decrease in the S-phase fraction. MMTV-v-Ha-ras mice were also interbred with MMTV-c-myc mice to determine whether ras/myc tumors, which possess high levels of spontaneous apoptosis, have the potential to regress through a further increase in apoptosis levels. The ras/myc tumors were found to respond nearly as efficiently to L-744,832 treatment as the MMTV-v-Ha-ras tumors, although no induction of apoptosis was observed. Rather, the tumor regression in the ras/myc mice was found to be mediated by a large reduction in the S-phase fraction. In contrast, treatment of transgenic mice harboring an activated MMTV-c-neu gene did not result in tumor regression. These results demonstrate that a farnesyltransferase inhibitor can induce regression of v-Ha-ras-bearing tumors by multiple mechanisms, including the activation of a suppressed apoptotic pathway, which is largely p53 independent, or by cell cycle alterations, depending upon the presence of various other oncogenic genetic alterations.

Differential regulation of cell cycle characteristics and apoptosis in MMTV-myc and MMTV-ras mouse mammary tumors.

We have used the MMTV-myc and MMTV-ras transgenic mouse mammary tumor models (T. A. Stewart et al., Cell, 38: 627-637, 1984, and E. Sinn et al., Cell, 49: 465-475, 1987) to evaluate how the c-myc and v-Ha-ras oncogenes influence tumor growth characteristics in vivo. MMTV-myc tumors had much higher levels of spontaneous apoptosis than MMTV-ras tumors, whereas intermediate levels were observed in MMTV-myc/ras tumors. Significant differences in cell cycle characteristics were also observed in tumors from mice of the three genotypes. Tumors from MMTV-myc mice had lower G1 and higher S-phase fractions than MMTV-ras tumors, with intermediate values again observed in the MMTV-myc/ras tumors. Despite these differences, however, tumor growth rates for the different groups were similar. These findings highlight the importance of the balance between cell cycle regulation and cell death in determining the kinetics of tumor growth and indicate that distinct oncogenes can have a profound influence on that balance.

Blocking the ZZ domain of sequestosome1/p62 suppresses myeloma growth and osteoclast formation in vitro and induces dramatic bone formation in myeloma-bearing bones in vivo.

We reported that p62 (sequestosome 1) serves as a signaling hub in bone marrow stromal cells (BMSCs) for the formation of signaling complexes, including NFκB, p38MAPK and JNK, that are involved in the increased osteoclastogenesis and multiple myeloma (MM) cell growth induced by BMSCs that are key contributors to multiple myeloma bone disease (MMBD), and demonstrated that the ZZ domain of p62 (p62-ZZ) is required for BMSC enhancement of MMBD. We recently identified a novel p62-ZZ inhibitor, XRK3F2, which inhibits MM cell growth and BMSC growth enhancement of human MM cells. In the current study, we evaluate the relative specificity of XRK3F2 for p62-ZZ, characterize XRK3F2's capacity to inhibit growth of primary MM cells and human MM cell lines, and test the in vivo effects of XRK3F2 in the immunocompetent 5TGM1 MM model. We found that XRK3F2 induces dramatic cortical bone formation that is restricted to MM containing bones and blocked the effects and upregulation of tumor necrosis factor alpha (TNFα), an osteoblast (OB) differentiation inhibitor that is increased in the MM bone marrow microenvironment and utilizes signaling complexes formed on p62-ZZ, in BMSC. Interestingly, XRK3F2 had no effect on non-MM bearing bone. These results demonstrate that targeting p62 in MM models has profound effects on MMBD.

Genetic determinants of response to chemotherapy in transgenic mouse mammary and salivary tumors.

Several transgenic mouse tumor models were utilized to explore how specific genetic alterations affect the tumor cell response to chemotherapeutic agents in vivo. Specifically, MMTV-ras transgenic mice were interbred to p53 knock-out mice to create a model for assessing the role of p53 in chemotherapeutic responses. In addition, MMTV-ras tumors were compared to MMTV-myc and MMTV-ras/myc tumors. Mice of each genotype reproducibly develop mammary and/or salivary tumors, but tumor growth dynamics vary considerably between genotypes. MMTV-ras/p53-/- tumors exhibit higher S phase fractions than MMTV-ras/p53+/+ tumors, although both tumor types display very low apoptosis levels. In contrast, MMTV-myc tumors exhibit both high S phase fractions and spontaneous apoptosis levels. Tumor-bearing mice of each genotype were treated with either doxorubicin or paclitaxel, and effects on overall tumor growth, cell cycle distribution and apoptosis were evaluated. Surprisingly, neither agent efficiently induced apoptosis in any of the tumor models, including those with wildtype p53. Rather, tumor responses were mediated primarily by changes in cell cycle distribution. However, the spontaneous apoptosis levels did serve as a predictor of tumor growth response, in that only those tumors with high pretreatment apoptosis levels underwent significant regression following treatment with either agent.

Ras protein farnesyltransferase: A strategic target for anticancer therapeutic development.

Ras proteins are guanine nucleotide-binding proteins that play pivotal roles in the control of normal and transformed cell growth and are among the most intensively studied proteins of the past decade. After stimulation by various growth factors and cytokines, Ras activates several downstream effectors, including the Raf-1/mitogen-activated protein kinase pathway and the Rac/Rho pathway. In approximately 30% of human cancers, including a substantial proportion of pancreatic and colon adenocarcinomas, mutated ras genes produce mutated proteins that remain locked in an active state, thereby relaying uncontrolled proliferative signals. Ras undergoes several posttranslational modifications that facilitate its attachment to the inner surface of the plasma membrane. The first-and most critical-modification is the addition of a farnesyl isoprenoid moiety in a reaction catalyzed by the enzyme protein farnesyltransferase (FTase). It follows that inhibiting FTase would prevent Ras from maturing into its biologically active form, and FTase is of considerable interest as a potential therapeutic target. Different classes of FTase inhibitors have been identified that block farnesylation of Ras, reverse Ras-mediated cell transformation in human cell lines, and inhibit the growth of human tumor cells in nude mice. In transgenic mice with established tumors, FTase inhibitors cause regression in some tumors, which appears to be mediated through both apoptosis and cell cycle regulation. FTase inhibitors have been well tolerated in animal studies and do not produce the generalized cytotoxic effects in normal tissues that are a major limitation of most conventional anticancer agents. There are ongoing clinical evaluations of FTase inhibitors to determine the feasibility of administering them on dose schedules like those that portend optimal therapeutic indices in preclinical studies. Because of the unique biologic aspects of FTase, designing disease-directed phase II and III evaluations of their effectiveness presents formidable challenges.

Cell lines of the pituitary gonadotrope lineage derived by targeted oncogenesis in transgenic mice.

Study of the molecular and cellular biology of the gonadotropin hormones would be greatly facilitated by the availability of immortalized anterior pituitary gonadotrope cell lines. We directed expression of the simian virus-40 (SV40) T-antigen (Tag) oncogene to specific cells in the anterior pituitary of transgenic mice using the promoter/enhancer region from the human glycoprotein hormone alpha-subunit gene. Transgenic mice carrying this fusion gene developed anterior pituitary tumors. Clonal cell lines established from these tumors express the endogenous mouse alpha-subunit gene and synthesize and secrete alpha-subunit protein. However, they do not express beta-subunit genes. Alpha-subunit mRNA is induced by GnRH in a dose- and time-dependent manner, but is not regulated by TRH. Thus, we have targeted tumorigenesis in transgenic mice to anterior pituitary cells of the gonadotrope lineage to immortalize this specific endocrine cell while maintaining several highly differentiated functions unique to gonadotropes.

Gonadotropin-releasing hormone receptor expression in Xenopus oocytes.

The rodent GnRH receptor was characterized in Xenopus oocytes injected with RNA isolated from rat pituitary and from a gonadotrope cell line, alpha T3, derived from a transgenic mouse. Three to 4 days after 150-200 ng RNA injection, 93% of the oocytes, which were recorded by voltage clamp, responded to 10(-7) M GnRH. The mean inward currents obtained after RNA injection were 620 +/- 88 nA (n = 22) with pituitary RNA and 1415 +/- 598 (n = 4) with alpha T3 RNA. The threshold GnRH concentration able to evoke the dose dependent current after pituitary RNA injection was 3 x 10(-9) M GnRH. The GnRH receptor response of the oocyte was antagonized by [D-Phe2,6,Pro3] GnRH and [N-Ac-D-Na](2)1, D-alpha D-Me, pCl-Phe2, D-Arg6, D-Ala10-NH2]GnRH and could be elicited by D-Ser(But)6,Pro9-N-ethylamide GnRH (buserelin). The reversal potential of the GnRH generated current as determined by voltage-ramp was -22.5 +/- 1.0 mV (n = 7) and -25.6 +/- 3.3 mV (n = 3) in pituitary and cell line RNA-injected oocytes respectively, consistent with the chloride reversal potential. The GnRH receptor response was virtually eliminated by intracellular EGTA injection but was unaffected by ligand application in calcium-free perfusate. The GnRH-evoked response is mimicked by intracellular injection of inositol 1,4,5-trisphosphate. To determine the size of the GnRH receptor mRNA, alpha T3 RNA was size fractionated through a sucrose gradient. The maximal GnRH response was induced by a fraction larger than the 28S ribosomal peak. Thus we find that oocytes injected with RNA from an appropriate source develop an electrophysiological response to GnRH which is dependent on intracellular calcium mobilization, is independent of extracellular calcium, and may be mediated by inositol 1,4,5-trisphosphate.

Intracellular responses to gonadotropin-releasing hormone in a clonal cell line of the gonadotrope lineage.

We recently derived a GnRH-responsive pituitary cell line of the gonadotrope lineage (alpha T3-1) by targeted oncogenesis in transgenic mice. Here, we report studies characterizing the GnRH receptors present in these cells and the intracellular responses to GnRH treatment. The receptors in alpha T3-1 cells show specificity for different GnRH analogs, with dissociation constants very similar to those found in normal rat and mouse pituitary. The concentration of receptors is within the range found in normal pituitary. The addition of GnRH or GnRH agonists increases phosphoinositide turnover and protein kinase-C translocation to membranes, and enhances activation of voltage-sensitive calcium channels. However, GnRH does not affect cAMP levels. Analysis of alpha-subunit mRNA levels demonstrated induction by GnRH and phorbol esters. Our results indicate that GnRH initiates a cascade of intracellular events that generate a set of second messengers, one or more of which is involved in the regulation of gene expression. The responses of alpha T3-1 cells to GnRH appear to have characteristics equivalent to those of primary pituitary gonadotropes, indicating the utility of this cell line as a model system for the study of GnRH responses.

Establishment of an osteocyte-like cell line, MLO-Y4.

Although osteocytes are the most abundant cells in bone, their functional role remains unclear. In part, this is due to lack of availability of osteocyte cell lines which can be studied in vitro. Since others have shown that cell lines can be readily developed from transgenic mice in which the SV40 large T-antigen oncogene is expressed under the control of a promoter which targets the cells of interest, we used this approach to develop an osteocyte cell line. We chose as a promoter osteocalcin, whose expression is essentially limited to bone cells and which is expressed more abundantly in osteocytes than in osteoblasts. From these transgenic mice, we isolated cells from the long bones using sequential collagenase digestion and maintained these cells on collagen-coated surfaces which are optimal for osteocyte maintenance and growth. We describe here the properties of a cell line cloned from these cultures, called MLO-Y4 (for murine long bone osteocyte Y4). The properties of MLO-Y4 cells are very similar to primary osteocytes. Like primary osteocytes and unlike primary osteoblasts, the cell line produces large amounts of osteocalcin but low amounts of alkaline phosphatase. The cells produce extensive, complex dendritic processes and are positive for T-antigen, for osteopontin, for the neural antigen CD44, and for connexin 43, a protein found in gap junctions. This cell line also produces very small amounts of type I collagen mRNA compared with primary osteoblasts. MLO-Y4 cells lack detectable mRNA for osteoblast-specific factor 2, which appears to be a positive marker for osteoblasts but may be a negative marker for osteocytes. This newly established cell line should prove useful for studying the effects of mechanical stress on osteocyte function and for determining the means whereby osteocytes communicate with other bone cells such as osteoblasts and osteoclasts.

Characterization of the mouse tartrate-resistant acid phosphatase (TRAP) gene promoter.

Tartrate-resistant acid phosphatase (TRAP) is an iron-binding protein that is highly expressed in osteoclasts. To characterize the regulation of TRAP gene expression, progressive 5' and 3' deletions of a 1.8 kb fragment containing the 5'-flanking sequence were fused to a luciferase reporter gene. Two nonoverlapping regions of this 1.8 kb fragment had promoter activity. The upstream promoter (P1) was located within the region from -881 bp to -463 bp relative to the ATG, while the downstream promoter (P2) was located between -363 bp to -1 bp in a region we have previously shown to be an intron in transcripts originating from the upstream promoter. A putative repressor region for the P2 promoter at -1846 bp to -1240 bp and a putative enhancer region at -962 bp to -881 bp relative to the ATG were identified. PCR analysis of promoter-specific transcription of the TRAP gene in various murine tissues showed that both promoters were active in several tissues. Transferrin-bound iron increased P1 promoter activity 2.5-fold and hemin decreased P1 promoter activity, but neither had any effect on P2 activity. These data show that the transcriptional regulation of the TRAP gene is complex and that iron may play a key role in TRAP gene regulation.

Cloning and characterization of the 5'-flanking region of the mouse tartrate-resistant acid phosphatase gene.

Little information is available on the molecular mechanisms controlling osteoclastic bone resorption. We used tartrate-resistant acid phosphatase (TRAP) to begin to investigate the regulation of bone resorption at the molecular level. TRAP is expressed at high levels in osteoclasts and may play an important role in the bone resorptive process. Therefore, we isolated the murine TRAP gene from a mouse spleen genomic library and characterized its promoter. A restriction map was generated for the 17 kb TRAP insert. A 2 kb SmaI fragment, containing the 5'-flanking region, was subcloned and the nucleotide sequence determined. Sequence analysis of the SmaI fragment revealed the presence of numerous candidate transcription factor binding sequences, including those for AP1 and H-APF-1. The H-APF-1 site matches the consensus sequence for the IL-6-regulated transcription factor. An intron was identified at -1 to -393 bp relative to the ATG. The presence of an intron was confirmed by PCR analysis of RNA isolated from murine osteoclasts. Primer extension analysis indicated the presence of a transcription initiation site at -552 bp from the ATG. The region from -1846 to 2bp relative to the ATG initiation codon drove the transient expression of a luciferase reporter gene when transfected into HRE H9 rabbit endometrial cells. PMA treatment of HRE H9 cells enhanced luciferase transcription approximately threefold. These data suggest that the TRAP promoter is complex and contains multiple regulatory elements. The availability of the TRAP promoter may also permit production of transgenic mice, which can be used to develop previously unavailable osteoclast cell lines.

Targeting simian virus 40 T antigen to the osteoclast in transgenic mice causes osteoclast tumors and transformation and apoptosis of osteoclasts.

Osteoclasts are terminally differentiated cells that express tartrate-resistant acid phosphatase (TRAP) at a higher level than other normal cells. Therefore, in an attempt to develop immortalized osteoclasts, we produced two lines of transgenic mice in which expression of the simian virus 40 T antigen oncogene was targeted to osteoclasts using the TRAP gene promoter. Osteoclasts were increased in number in bones from both lines. More than 50% of them appeared morphologically transformed, 2-5% were mitotic, but, unexpectedly, 5% were apoptotic. Osteoclast tumors were observed occasionally in one line of mice (line 4), and sheets of TRAP-positive cells (tumorlets) developed in most mice in both lines. Although cells isolated from these tumorlets formed multinucleated TRAP-positive cells that resorbed bone in vitro, to date we have been unable to develop an immortalized osteoclast cell line from them. Osteoclasts from one line (line 5) had reduced ruffled border formation and a higher level of T-antigen expression than osteoclasts in the other line (line 4), and these features were associated with the presence of osteopetrosis. However, osteoclasts from these osteopetrotic mice and from line 4 mice resorbed bone normally when the mice were treated with interleukin-1. These findings indicate that T antigen can be targeted to osteoclasts in transgenic mice and causes osteoclast transformation, tumors, mitosis, and apoptosis. When T antigen is expressed at high levels, functional impairment of osteoclasts can be detected. Furthermore, these results suggest that T antigen is insufficient on its own to immortalize cells in the osteoclast lineage.

Osteoclasts formed by measles virus-infected osteoclast precursors from hCD46 transgenic mice express characteristics of pagetic osteoclasts.

Pagetic osteoclasts (OCLs) are abnormal in size and contain paramyxoviral-like nuclear inclusions that cross-react with antibodies to measles virus (MV). However, the role that MV infection plays in Paget's disease is unknown, because no animal model of Paget's disease is available. Therefore, we targeted a cellular MV receptor, human CD46 (hCD46), to cells in the OCL lineage in transgenic mice using the mouse tartrate-resistant acid phosphatase (TRAP) gene promoter. In vitro infection of OCL precursors from hCD46 transgenic mice with MV significantly increased OCL formation in bone marrow cultures. The numbers of TRAP-positive mononuclear cells and CFU-GM, the earliest identifiable OCL precursor, were also significantly increased. MV-infected OCLs formed from hCD46 marrow were increased in size, contained markedly increased numbers of nuclei, and had increased bone-resorbing capacity per OCL compared with OCLs formed from marrow of nontransgenic littermates. Furthermore, IL-6 and 24-hydroxylase messenger RNA expression levels were increased in MV-infected hCD46 transgenic mouse bone marrow cultures. Treatment of MV-infected hCD46 marrow cultures with a neutralizing antibody to IL-6 blocked the increased OCL formation seen in these cultures. These data demonstrate that MV infection of OCL precursors results in OCLs that have many features of pagetic OCLs, that the enhanced OCL formation is in part mediated by increased IL-6 expression induced by MV infection, and suggest that the hCD46 transgenic mouse may be a useful model for examining the effects of MV infection on OCL formation in vivo.

Immortalized murine osteoblasts derived from BMP 2-T-antigen expressing transgenic mice.

Osteoblast cell lines capable of undergoing bone formation in vitro would provide useful models for understanding gene expression during bone cell differentiation. To that end, transgenic mice were produced using a 2.9-kilobase bone morphogenetic protein 2 (BMP-2) promoter fragment, driving simian virus 40 T antigen as the transgene. The expression of simian virus 40 T antigen driven by the BMP-2 promoter immortalizes the cells. From the calvaria of the transgenic mouse, several osteoblastic cell lines were isolated and cloned. One clonal osteoblast cell line, called 2T3, has been characterized and shown to produce mineralized bone nodules. Recombinant human BMP-2 (rhBMP-2) accelerates the formation of these mineralized bone nodules. 2T3 cells express alkaline phosphatase, collagen type I, osteocalcin, and endogenous BMP-2 messenger RNA (mRNA) in a similar chronological order as normal freshly isolated fetal rat calvarial cells during early nodule formation and subsequent mineralization. The 2T3 cells also exhibit extensive growth and multilayering during differentiation, as demonstrated by growth curves and transmission electron microscopy. As with freshly isolated fetal rat calvarial cells, 1,25-dihydroxyvitamin D3 inhibited alkaline phosphatase activity and alkaline phosphatase mRNA expression, but stimulated osteocalcin mRNA expression, but stimulated osteocalcin mRNA expression. rhBMP-2 also accelerated the expression of alkaline phosphatase activity and mRNA, osteocalcin mRNA, and BMP-2 mRNA in 2T3 cells along with the formation of larger and more mineralized bone nodules. The 2T3 cell exhibits autoregulation at the mRNA and transcriptional levels. The 2T3 osteoblast cell line offers a system for examining autoregulation of the BMP-2 gene and downstream gene expression during osteoblast differentiation. 2T3 cells are reclonable and maintain their differentiation capabilities.