Overexpression of Ad5 precursor terminal protein accelerates recombinant adenovirus packaging and amplification in HEK-293 packaging cells.

Recombinant adenoviruses are one of the most common vehicles for efficient in vitro and in vivo gene deliveries. Here, we investigate whether exogenous precursor terminal protein (pTP) expression in 293 cells improves the efficiency of adenovirus packaging and amplification. We used a piggyBac transposon-based vector and engineered a stable 293 line that expresses high level of Ad5 pTP, designated as 293pTP. Using the AdBMP6-GLuc that expresses green fluorescent protein (GFP), BMP6 and Gaussia luciferase, we found that the infectivity of AdBMP6-GLuc viral samples packaged in 293pTP cells was titrated up to 19.3 times higher than that packaged in parental 293 cells. AdBMP6-GLuc viral samples packaged in 293pTP cells exhibited significantly higher transduction efficiency in 143B and immortalized mouse embryonic fibroblast (iMEF) cells, as assessed by fluorescence-activated cell sorting analysis of GFP-positive cells, the luciferase activity assay and BMP6-induced osteogenic marker alkaline phosphatase activities in iMEFs. When adenovirus amplification efficiency was analyzed, we found that 293pTP cells infected with AdBMP6-GLuc yielded up to 12.6 times higher titer than that in parental 293 cells, especially at lower multiplicities of infection. These results strongly suggest that exogenous pTP expression may accelerate the packaging and amplification of recombinant adenoviruses. Thus, the engineered 293pTP cells should be a superior packaging line for efficient adenovirus production.

Insulin-like growth factor binding protein 5 suppresses tumor growth and metastasis of human osteosarcoma.

Osteosarcoma (OS) is the most common primary malignancy of bone. There is a critical need to identify the events that lead to the poorly understood mechanism of OS development and metastasis. The goal of this investigation is to identify and characterize a novel marker of OS progression. We have established and characterized a highly metastatic OS subline that is derived from the less metastatic human MG63 line through serial passages in nude mice via intratibial injections. Microarray analysis of the parental MG63, the highly metastatic MG63.2 subline, as well as the corresponding primary tumors and pulmonary metastases revealed insulin-like growth factor binding protein 5 (IGFBP5) to be one of the significantly downregulated genes in the metastatic subline. Confirmatory quantitative RT-PCR on 20 genes of interest demonstrated IGFBP5 to be the most differentially expressed and was therefore chosen to be one of the genes for further investigation. Adenoviral mediated overexpression and knockdown of IGFBP5 in the MG63 and MG63.2 cell lines, as well as other OS lines (143B and MNNG/HOS) that are independent of our MG63 lines, were employed to examine the role of IGFBP5. We found that overexpression of IGFBP5 inhibited in vitro cell proliferation, migration and invasion of OS cells. Additionally, IGFBP5 overexpression promoted apoptosis and cell cycle arrest in the G1 phase. In an orthotopic xenograft animal model, overexpression of IGFBP5 inhibited OS tumor growth and pulmonary metastases. Conversely, siRNA-mediated knockdown of IGFBP5 promoted OS tumor growth and pulmonary metastases in vivo. Immunohistochemical staining of patient-matched primary and metastatic OS samples demonstrated decreased IGFBP5 expression in the metastases. These results suggest 1) a role for IGFBP5 as a novel marker that has an important role in the pathogenesis of OS, and 2) that the loss of IGFBP5 function may contribute to more metastatic phenotypes in OS.

Characterization of the distinct orthotopic bone-forming activity of 14 BMPs using recombinant adenovirus-mediated gene delivery.

Efficacious bone regeneration could revolutionize the clinical management of bone and musculoskeletal disorders. Although several bone morphogenetic proteins (BMPs) (mostly BMP-2 and BMP-7) have been shown to induce bone formation, it is unclear whether the currently used BMPs represent the most osteogenic ones. Until recently, comprehensive analysis of osteogenic activity of all BMPs has been hampered by the fact that recombinant proteins are either not biologically active or not available for all BMPs. In this study, we used recombinant adenoviruses expressing the 14 types of BMPs (AdBMPs), and demonstrated that, in addition to currently used BMP-2 and BMP-7, BMP-6 and BMP-9 effectively induced orthotopic ossification when either AdBMP-transduced osteoblast progenitors or the viral vectors were injected into the quadriceps of athymic mice. Radiographic and histological evaluation demonstrated that BMP-6 and BMP-9 induced the most robust and mature ossification at multiple time points. BMP-3, a negative regulator of bone formation, was shown to effectively inhibit orthotopic ossification induced by BMP-2, BMP-6, and BMP-7. However, BMP-3 exerted no inhibitory effect on BMP-9-induced bone formation, suggesting that BMP-9 may transduce osteogenic signaling differently. Our findings suggest that BMP-6 and BMP-9 may represent more effective osteogenic factors for bone regeneration.

The unsatisfactory margin in breast cancer surgery.

BACKGROUND: Surgical margin involvement with breast cancer usually results in obligatory reexcision or mastectomy. While unalterable occult host and pathologic factors may interfere with margin clearance during the initial excision, it is possible that alterations in surgical technique might increase the likelihood of obtaining satisfactory margins. METHODS: Two hundred and thirty-five patients who were candidates for breast conservation therapy were identified for 1991 and 1996 using the Tumor Registry. Margins were defined as "unsatisfactory" if there was microscopic involvement with tumor or the margin was close at initial excisional biopsy and the surgeon opted for reexcision. Multiple logistic regression analyses of factors associated with margin status were performed. RESULTS: One hundred thirty-two (56%) patients had positive or close (unsatisfactory) margins; this rate increased from 51% in 1991 to 59% in 1996. Patients with unsatisfactory margins underwent more procedures (mean 2.0 versus 1.2; P <0.0001) than patients whose margins were satisfactory. The breast conservation rate for patients with unsatisfactory margins was 64% compared with 99% for patients with satisfactory margins. A multiple logistic regression demonstrated that patients with unsatisfactory margins were 67 times more likely to have a mastectomy than patients whose margins were satisfactory after adjusting for other significant factors (P <0.0001). The practice of fine needle aspiration biopsy, orientation of specimen margins by the surgeon, and reexcision of tumor at the first operation were statistically significant technical factors in obtaining satisfactory margins. Significant pathology factors were extensive intraductal component (EIC), lobular or ductal extension, and tumor size. CONCLUSION: These data show that technical factors in the surgical management of breast cancer, as well as biological factors such as EIC, can influence the success of breast conservation.

Chromosome 17-mediated dormancy of AT6.1 prostate cancer micrometastases.

To improve the diagnosis and treatment of cancer, an increased understanding of the molecular and cellular changes that regulate metastatic ability is required. We have recently demonstrated a prostate cancer metastasis-suppressor activity encoded by a discontinuous approximately 70-cM region of human chromosome. The presence of this region suppresses the spontaneous metastatic ability of AT6.1 rat prostatic cancer cells by greater than 30-fold (M. A. Chekmareva et al., Prostate, 33: 271-280, 1997). Interestingly, a number of potentially important genes which have been mapped to human chromosome 17, including TP53, NM23, and BRCA1, are not retained (M. A. Chekmareva et al., cited above) or are not expressed in these microcell hybrids (B. A. Yoshida et al., In Vivo, in press), which suggests the presence of a novel metastasis-suppressor gene(s) or novel function of a known gene(s) encoded by this region(s). We hypothesize that identification of the "step" in the metastatic cascade that is inhibited by the presence of the approximately 70-cM metastasis-suppressor region will facilitate the identification of candidate metastasis-suppressor genes. For a cancer cell to metastasize, it must escape from the primary tumor, enter the circulation, arrest in the microcirculation, extravasate into a tissue compartment, and grow. This suppression of spontaneous macroscopic lung metastases could be due to the inhibition of a number of steps within this cascade. Results of the current study demonstrate that AT6.1 cells containing the approximately 70-cM region (AT6.1-17-4 cells) escape from the primary tumor and arrest in the lung but are growth-inhibited unless the metastasis-suppressor region is lost. This growth inhibition seems to result from an effect of one or more genes at the metastatic site and not from a circulating angiogenesis inhibitor. Our findings suggest that the approximately 70-cM region of human chromosome 17 may encode a gene(s) that regulates the "dormancy" of AT6.1-17-4 micrometastases.

Identification of a novel metastasis-suppressor region on human chromosome 12.

There is a critical need for markers that can be used to predict accurately the malignant potential of histological prostate cancers (J. T. Isaacs. Am. J. Pathol., 150: 1511-1521, 1997). Metastasis-suppressor genes are attractive candidates for marker development because, by definition, their loss should be associated with the acquisition of metastatic ability. In an effort to identify such genes, a single copy of human chromosome 12, tagged with the neomycin resistance gene, was introduced into highly metastatic Dunning AT6.1 prostate cancer cells by microcell-mediated chromosomal transfer. Thirty-two AT6.1-12 clonal cell lines were established and the region(s) of chromosome 12 retained was determined by sequence tagged site-based PCR analysis. Representative AT6.1-12 clones containing overlapping regions of chromosome 12 were characterized cytogenetically and were shown to have a normal complement of parental AT6.1 rat chromosomes. Fluorescence in situ hybridization, performed on representative AT6.1-12 hybrids, demonstrated a single human chromosome 12-specific signal. The metastatic ability of six representative clones was tested in immunodeficient mice. All of the AT6.1-12 clones showed the same in vivo growth rates as the control AT6.1-neo cells. Clonal cell lines that contained a conserved approximately 70-cM portion of chromosome 12 (e.g., AT6.1-12-8, -8-1, and -8-3), showed a >30-fold suppression in the number of macroscopic surface lung metastases. Mice that received injections of these cells developed a mean number 4 lung metastases whereas mice that received injections of other AT6.1-12 hybrids (lacking the approximately 70-cM region) or AT6.1-neo control cells, developed a mean number of 140 metastases. Interestingly, histological examination of the lungs of the mice that received injections of AT6.1-12-8 cells showed essentially no microscopic metastases. These findings suggest that a gene(s) encoded by the approximately 70-cM portion of human chromosome 12 suppresses an early step in the metastatic cascade.

Posttranslational regulation of plakoglobin expression. Influence of the desmosomal cadherins on plakoglobin metabolic stability.

Desmosomes are adhesive intercellular junctions that act as cell surface attachment sites for intermediate filaments. The desmosomal glycoproteins, desmogleins and desmocollins, are members of the cadherin family of adhesion molecules. In addition, desmoglein has been shown to coimmunoprecipitate with the junctional protein plakoglobin. To characterize further the interaction between plakoglobin and the desmosomal cadherins, stable mouse fibroblast (L-cells) cell lines were generated that express plakoglobin, desmoglein and plakoglobin, or desmocollin and plakoglobin. L-cell lines transfected with a plasmid encoding human plakoglobin expressed plakoglobin mRNA but very little plakoglobin protein. However, plakoglobin protein was expressed at high levels in L-cells coexpressing either desmoglein or desmocollin. In addition, both desmocollin and desmoglein were found to coimmunoprecipitate with plakoglobin. The transient expression of desmoglein in L-cell lines expressing plakoglobin mRNA resulted in the formation of a complex between plakoglobin and desmoglein and in the accumulation of plakoglobin protein. Furthermore, the rate of plakoglobin protein degradation was decreased by 15-20-fold in cell lines expressing either desmoglein or desmocollin. These results demonstrate that the desmosomal cadherins posttranslationally regulate plakoglobin expression by decreasing the rate of plakoglobin degradation.

Functional analysis of desmoplakin domains: specification of the interaction with keratin versus vimentin intermediate filament networks.

We previously demonstrated that truncated desmoplakin I (DP I) molecules containing the carboxyl terminus specifically coalign with and disrupt both keratin and vimentin intermediate filament (IF) networks when overexpressed in tissue culture cells (Stappenbeck, T. S., and K. J. Green. J. Cell Biol. 116:1197-1209). These experiments suggested that the DP carboxyl-terminal domain is involved either directly or indirectly in linking IF with the desmosome. Using a similar approach, we have now investigated the behavior of ectopically expressed full-length DP I in cultured cells. In addition, we have further dissected the functional sequences in the carboxyl terminus of DP I that facilitate the interaction with IF networks. Transient transfection of a clone encoding full-length DP I into COS-7 cells produced protein that appeared in some cells to associate with desmosomes and in others to coalign with and disrupt IF. Deletion of the carboxyl terminus from this clone resulted in protein that still appeared capable of associating with desmosomes but not interacting with IF networks. As the amino terminus appeared to be dispensable for IF interaction, we made finer deletions in the carboxyl terminus of DP based on blocks of sequence similarity with the related molecules bullous pemphigoid antigen and plectin. We found a sequence at the very carboxyl terminus of DP that was necessary for coalignment with and disruption of keratin IF but not vimentin IF. Furthermore, the coalignment of specific DP proteins along keratin IF but not vimentin IF was correlated with resistance to extraction by Triton. The striking uncoupling resulting from the deletion of specific DP sequences suggests that the carboxyl terminus of DP interacts differentially with keratin and vimentin IF networks.