PPAR␥ and C/EBP␣ are critical transcription factors in adipogenesis, but the precise role of these proteins has been difficult to ascertain because they positively regulate each other's expression. Questions remain about whether these factors operate independently in separate, parallel pathways of differentiation, or whether a single pathway exists. PPAR␥ can promote adipogenesis in C/EBP␣-deficient cells, but the converse has not been tested. We have created an immortalized line of fibroblasts lacking PPAR␥, which we use to show that C/EBP␣ has no ability to promote adipogenesis in the absence of PPAR␥. These results indicate that C/EBP␣ and PPAR␥ participate in a single pathway of fat cell development with PPAR␥ being the proximal effector of adipogenesis. Received September 27, 2001; revised version accepted November 9, 2001. Adipogenesis is the process by which undifferentiated precursor cells differentiate into fat cells. This has become one of the most intensively studied developmental processes for at least two reasons: the increasing prevalence of obesity in our society has focused attention on many aspects of fat cell biology, and the availability of good cell culture models of adipocyte differentiation has permitted detailed studies not possible in other systems. Experiments using these in vitro models of adipogenesis, which include the 3T3-L1 and 3T3-F442A lines, have illustrated the transcriptional cascade that promotes fat cell differentiation (Rosen et al. 2000). Representatives of several transcription factor families have been implicated in this process, including the CCAAT/enhancer binding proteins C/EBP␣, C/EBP␤, and C/EBP␦; the nuclear hormone receptor peroxisome proliferator-activated receptor ␥ (PPAR␥); and the basic helix-loop-helix protein ADD1/SREBP1c. Studies in adipogenic cell lines have shown that hormonal induction of differentiation is rapidly followed by expression of C/EBP␤ and C/EBP␦ (Cao et al. 1991;Yeh et al. 1995). Within the next day or so, levels of these proteins peak and then begin to drift downward, coincident with a rise in C/EBP␣ and PPAR␥. These latter factors induce gene expression changes characteristic of mature adipocytes and remain elevated for the life of the cell. In the present model of the transcriptional cascade leading to adipogenesis, C/EBP␤ and C/EBP␦ induce low levels of PPAR␥ and C/EBP␣, which are then able to induce each other's expression in a positive feedback loop that promotes and maintains the differentiated state. This model is consistent with gain-offunction data showing that the addition of either PPAR␥ or C/EBP␣ can promote adipogenesis in fibroblast cell lines (Lin and Lane 1994;Tontonoz et al. 1994).Loss-of-function studies have shown convincingly that PPAR␥ is required for adipogenesis in vivo and in vitro, and cells lacking PPAR␥ express greatly reduced levels of C/EBP␣ (Barak et al. 1999;Kubota et al. 1999;Rosen et al. 1999). Similarly, fibroblasts lacking C/EBP␣ have reduced adipogenic potential, and express reduced levels of PPAR␥ (Wu...

Antibody-drug conjugate (ADC) which delivers cytotoxic drugs specifically into targeted cells through internalization and lysosomal trafficking has emerged as an effective cancer therapy. We show that a bivalent biparatopic antibody targeting two non-overlapping epitopes on HER2 can induce HER2 receptor clustering, which in turn promotes robust internalization, lysosomal trafficking, and degradation. When conjugated with a tubulysin-based microtubule inhibitor, the biparatopic ADC demonstrates superior anti-tumor activity over ado-trastuzumab emtansine (T-DM1) in tumor models representing various patient subpopulations, including T-DM1 eligible, T-DM1 ineligible, and T-DM1 relapsed/refractory. Our findings indicate that this biparatopic ADC has promising potential as an effective therapy for metastatic breast cancer and a broader patient population may benefit from this unique HER2-targeting ADC.

No abstract

We previously showed that a single intrapleural dose of an adenoviral vector expressing interferon-beta (Ad.IFN-beta) in patients with malignant pleural mesothelioma (MPM) or malignant pleural effusions (MPE) resulted in gene transfer, humoral antitumor immune responses, and anecdotal clinical responses manifested by modified Response Evaluation Criteria in Solid Tumors (RECIST) disease stability in 3 of 10 patients at 2 months and an additional patient with significant metabolic response on positron emission tomography (PET) imaging. This phase I trial was conducted to determine whether using two doses of Ad.IFN-beta vector would be superior. Ten patients with MPM and seven with MPE received two doses of Ad.IFN-beta through an indwelling pleural catheter. Repeated doses were generally well tolerated. High levels of IFN-beta were detected in pleural fluid after the first dose; however, only minimal levels were seen after the second dose of vector. Lack of expression correlated with the rapid induction of neutralizing Ad antibodies (Nabs). Antibody responses against tumor antigens were induced in most patients. At 2 months, modified RECIST responses were as follows: one partial response, two stable disease, nine progressive disease, and two nonmeasurable disease. One patient died after 1 month. By PET scanning, 2 patients had mixed responses and 11 had stable disease. There were seven patients with survival times longer than 18 months. This approach was safe, induced immune responses and disease stability. However, rapid development of Nabs prevented effective gene transfer after the second dose, even with a dose interval as short as 7 days.

Mü llerian inhibiting substance (MIS) is a key element required to complete mammalian male sex differentiation. The expression pattern of MIS is tightly regulated in fetal, neonatal, and prepubertal testes and adult ovaries and is well conserved among mammalian species. Although several factors have been shown to be essential to MIS expression, its regulatory mechanisms are not fully understood. We have examined MIS promoter activity in 2-day postnatal primary cultures of rat Sertoli cells that continue to express endogenous MIS mRNA. Using this system, we found that the region between human MIS؊269 and ؊192 is necessary for full MIS promoter activity. We identified by DNase I footprint and electrophoretic mobility-shift analyses a distal steroidogenic factor-1 (SF-1)-binding site that is essential for full promoter activity. Mutational analysis of this new distal SF-1 site and the previously identified proximal SF-1 site showed that both are necessary for transcriptional activation. Moreover, the proximal promoter also contains multiple GATA-4-binding sites that are essential for functional promoter activity. Thus multiple SF-1-and GATA-4-binding sites in the MIS promoter are required for normal tissue-specific and developmental expression of MIS.Mü llerian inhibiting substance promoter M üllerian inhibiting substance (MIS), also called antiMüllerian hormone, a glycoprotein homodimer belonging to the transforming growth factor ␤ superfamily, is a critical component of sex differentiation responsible for regression in the male embryo of the Müllerian ducts, which in a normal female embryo become the uterus, fallopian tubes, and upper vagina (1). In the rat, MIS is expressed in fetal Sertoli cells from the time of testis differentiation (13 days postcoitum) (2). Both MIS mRNA and protein remain high after birth and fall precipitously after day 5 to a low level, where they remain throughout adult life. Conversely, MIS mRNA is undetectable in the fetal ovaries but becomes increasingly expressed after birth in the granulosa cells of developing follicles (3-5).The complex expression pattern of MIS suggests that it is tightly regulated. The MIS genes from mouse (6), rat (7), bovine (8), porcine (9), chicken (10), and human (8, 11) have been cloned, and all mammalian proximal promoters show regions of evolutionary conservation (Fig. 1). The mouse MIS transcriptional start site is located only 328 bp downstream of the SAP62 gene, suggesting that the region conferring critical regulation of MIS expression is located within close proximity of the transcriptional start site (12).Several factors important for sex determination have been proposed to regulate MIS expression, including SRY (13), SOX9 (14, 15), SF-1 (16, 17), WT-1 (18, 19), Dax-1 (19), and 21), and all except Dax-1 and possibly WT-1 can bind to the Ϫ180 bp region upstream of the transcriptional start site of MIS. Moreover, evidence is convincing that transcriptional upregulation of MIS requires coordinate interactions between SF-1 and SOX9 (15), GATA-4 (21), an...

Metastatic disease is the major cause of morbidity and mortality in cancer. Although surgery, chemotherapy, or radiation can often control primary tumor growth, successful eradication of disseminated metastases remains rare. We have now tested whether direct targeting tumor tissues to generate antitumor immune response before surgical excision produces sufficient CTL against micrometastases. One unsolved problem is whether such response allows coming CTL to be educated and then exit the tumor site. Another unsolved problem is whether these CTL can then patrol and effectively eliminate spontaneously metastasized tumor cells in the periphery. In this study, we have shown that adenovirus-expressing TNFSF14 [LIGHT (name derived from homologous to lymphotoxins, shows inducible expression, and competes with herpes simplex virus glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes); Ad-LIGHT] inoculated directly into primary 4T1 tumor, a highly aggressive, spontaneously metastasizing mammary carcinoma, followed by surgical removal of the primary tumor can eradicate established and disseminated metastatic tumor cells in the peripheral tissues. Furthermore, we clearly show with a fibrosarcoma model Ag104Ld that local treatment can generate plenty of tumor-specific CTL that exit the primary tumor and infiltrate distal tumors to completely eradicate distal tumors. Therefore, targeting the primary tumor with Ad-LIGHT before surgical excision is a new strategy to elicit better immune response for the eradication of spontaneous metastases.

The binding of growth hormone (GH) to its receptor results in its dimerization followed by activation of Jak2 kinase and tyrosine phosphorylation of the GH receptor itself, as well as Jak2 and the transcription factors Stat1, -3, and -5. In order to study the role of GH receptor tyrosine phosphorylation in intracellular signaling, we constructed GH receptors in which combinations of tyrosines were mutated to phenylalanines. We identified three tyrosine residues at positions 534, 566, and 627 that were required for activation of GH-stimulated transcription of the serine protease inhibitor (Spi) 2.1 promoter. Any of these three tyrosines is able to independently mediate GH-induced transcription, indicating redundancy in this part of the GH receptor. Tyrosine phosphorylation was not required for GH stimulation of mitogen-activated protein (MAP) kinase activity or for GH-stimulated Ca 2؉ channel activation since these pathways were normal in cells expressing a GH receptor in which all eight intracellular tyrosines were mutated to phenylalanines. Activation of Stat5 by GH was, however, abolished in cells expressing the GH receptor lacking intracellular tyrosines. This study demonstrates that specific tyrosines in the GH receptor are required for transcriptional signaling possibly by their role in the activation of transcription factor Stat5.Pituitary growth hormone (GH) 1 is the major regulator of postnatal growth (1). The actions of GH at the cellular level include direct mitogenic effects (2, 3), insulin-like and insulinantagonizing metabolic effects (4), as well as gene regulatory actions (5-7). All of these effects are initiated by the binding of GH to its receptor, which belongs to the cytokine receptor superfamily. Members of this family of receptors activate cytoplasmic tyrosine kinases of the Jak family, and these activated kinases are required for most receptor-initiated signaling pathways (8). The activated Jak2 kinase has been shown to phosphorylate several intracellular substrates including the GH receptor itself, as well as transcription factors of the Stat family (9 -11).Not only does GH activate the Jak/Stat pathway in which specific tyrosine phosphorylation of Stat1, -3, and -5 occurs in response to GH, resulting in dimerization, nuclear translocation, and binding to ␥-interferon activated sequence-like elements (11-13), but we and others have previously identified several alternative signaling pathways induced by the activated GH receptor. Stimulation and tyrosine phosphorylation of MAP kinase by GH have been studied both in cultured cells (14,15) and in vivo (16). In addition to activation of MAP kinase, its translocation to the nucleus has also been demonstrated. The activation of MAP kinase by GH is dependent upon the proline-rich box 1 domain of the GH receptor that presumably is directly involved in the binding of Jak2. When the box 1 domain is deleted or the prolines are mutated to alanines, the GH receptor is no longer able to mediate GHinduced MAP kinase activity. The functional role of MAP k...

Apocytochrome c is synthesized in the cytoplasm, transported to the mitochondrial intermembrane space, and subsequently covalently attached to heme in a reaction catalyzed by the enzyme cytochrome c heme lyase. We have investigated the amino acid sequences in cytochrome c which are required for mitochondrial import, using a systematic series of site-directed alterations of the CYC7-H3 gene which encodes iso-2-cytochrome c in the yeast Saccharomyces cerevisiae. Import of the altered apocytochromes c was assayed in yeast strains that overexpressed cytochrome c heme lyase. Under these conditions, there was efficient mitochondrial accumulation of forms of apocytochrome c which are incapable of having heme covalently attached. In fact, all apocytochromes c containing deletions located to the carboxyl-terminal side of His27 efficiently accumulated in the mitochondria of strains overexpressing heme lyase, even though all but one of these deletion-containing proteins were incapable of heme attachment. A minimum length of polypeptide chain at the extreme amino terminus of cytochrome c, rather than any specific sequence element in this region, appears to be required for efficient mitochondrial import. Certain amino acid substitutions in the region extending from Gly15 to Leu18, at residue Phe19 and at residue His27, lead to reduced mitochondrial import of apocytochrome c, resulting from stalling of the altered apocytochrome c in partially imported states.