Synergistic cytotoxicity of radiation and oncolytic Lister strain vaccinia in (V600D/E)BRAF mutant melanoma depends on JNK and TNF-α signaling.

Melanoma is an aggressive skin cancer that carries an extremely poor prognosis when local invasion, nodal spread or systemic metastasis has occurred. Recent advances in melanoma biology have revealed that RAS-RAF-MEK-ERK signaling has a pivotal role in governing disease progression and treatment resistance. Proof-of-concept clinical studies have shown that direct BRAF inhibition yields impressive responses in advanced disease but these are short-lived as treatment resistance rapidly emerges. Therefore, there is a pressing need to develop new targeted strategies for BRAF mutant melanoma. As such, oncolytic viruses represent a promising cancer-specific approach with significant activity in melanoma. This study investigated interactions between genetically-modified vaccinia virus (GLV-1h68) and radiotherapy in melanoma cell lines with BRAF mutant, Ras mutant or wild-type genotype. Preclinical studies revealed that GLV-1h68 combined with radiotherapy significantly increased cytotoxicity and apoptosis relative to either single agent in (V600D)BRAF/(V600E)BRAF mutant melanoma in vitro and in vivo. The mechanism of enhanced cytotoxicity with GLV-1h68/radiation (RT) was independent of viral replication and due to attenuation of JNK, p38 and ERK MAPK phosphorylation specifically in BRAF mutant cells. Further studies showed that JNK pathway inhibition sensitized BRAF mutant cells to GLV-1h68-mediated cell death, mimicking the effect of RT. GLV-1h68 infection activated MAPK signaling in (V600D)BRAF/(V600E)BRAF mutant cell lines and this was associated with TNF-α secretion which, in turn, provided a prosurvival signal. Combination GLV-1h68/RT (or GLV-1h68/JNK inhibition) caused abrogation of TNF-α secretion. These data provide a strong rationale for combining GLV-1h68 with irradiation in (V600D/E)BRAF mutant tumors.

Oncolytic vaccinia virus in combination with radiation shows synergistic antitumor efficacy in pancreatic cancer.

Combining oncolytic viruses with conventional therapy such as radiation is an innovative option for pancreatic cancer. We demonstrated that combination of GLV-1h151 and radiation yielded a synergistic cytotoxic effect, with the greatest effect achieved in the AsPC-1cell line. Combination treatment significantly increased apoptosis compared with either single treatment or the control group. In mice bearing human pancreatic tumor xenografts, combination treatment resulted in significantly enhanced inhibition of tumor growth. No evidence of toxicity was observed in mice. These results indicate that the combination of GLV-1h151 and radiation has great potential for translation into clinic practice.

[Significance of autopsy in patients with head and neck cancer]. / Stellenwert der Autopsie bei Patienten mit Kopf-Halstumoren.

BACKGROUND: Nowadays, the morphological assessment of samples obtained from living patients has a greater importance than the scientific knowledge which is gained by autopsy. Therefore, the aim of the study was a retrospective analysis of causes of death in patients with head and neck cancer. MATERIAL AND METHODS: The autopsy rate, clinical parameters of oncologic patients as well as autopsy findings like lethal complications, distant metastases and second primary tumors were retrospectively analyzed. RESULTS: From 1968 to 2007 in 91 patients with malignant tumors of the head and neck an autopsy was performed. In these 39 years an autopsy was performed in 45.9% of dead oncologic patients. Autopsy findings revealed distant metastases in 46.2% and second primary tumors in 17.6% of the patients. 49.5% of the patients died from pneumonia, 20.9% from tumor bleeding and 10% from progressive cachexia. CONCLUSION: The study confirms the global trend of a decline in autopsy numbers in the last 3 decades. However, as an important instrument of quality assurance autopsies continue to play an essential and indispensable role in medical research.

Remodelling of the palatal dome following rapid maxillary expansion (RME): laser scan-quantifications during a low growth period.

OBJECTIVES: To evaluate changes in the palatal vault after rapid maxillary expansion (RME) with bonded splint appliances. SETTING AND SAMPLE POPULATION: The sample comprised 24 children (12 boys and 12 girls) with mixed dentition (mean age 8.3 years; range 6.4-10.4 years). MATERIALS AND METHODS: Following expansion, the splint appliance was used as a retainer for 6 months and then removed. Study casts were taken before RME (T0) and when the appliance was removed (T1). Then, 3D laser scans were taken to build complete 3D jaw models. Frontal cross sections were constructed at 53-63, 55-65 and 16-26, exported as coordinates, and finite element calculated to quantify their area, width and height. Maxillary length was also determined. RESULTS: Paired t-tests indicated statistically significant increases in the average palatal width (T1-T0=6.53-6.79 mm) and cross-sectional area (T1-T0=20.39-21.39 mm2) after RME (p<0.001). However, small but statistically significant reductions were observed in palatal height (T1-T0=-0.49 mm, only at 55-65; p<0.001) and length (T1-T0=-0.54 mm; p<0.01). Linear regression analysis showed statistically significant (p<0.001) direct correlations between the widths and respective cross-sectional areas. Age did not influence any measurement. The reliability of the measurements was examined with an intraclass correlation coefficient (ICC). We found an ICC>0.99 (p<0.001) for all tested parameters. CONCLUSIONS: Rapid maxillary expansion distinctly increased mean palatal widths and cross-sectional areas. However, palatal height (55-65) and maxillary length decreased to a small extent.

Turbulence Visualization at the Terascale on Desktop PCs.

Despite the ongoing efforts in turbulence research, the universal properties of the turbulence small-scale structure and the relationships between small- and large-scale turbulent motions are not yet fully understood. The visually guided exploration of turbulence features, including the interactive selection and simultaneous visualization of multiple features, can further progress our understanding of turbulence. Accomplishing this task for flow fields in which the full turbulence spectrum is well resolved is challenging on desktop computers. This is due to the extreme resolution of such fields, requiring memory and bandwidth capacities going beyond what is currently available. To overcome these limitations, we present a GPU system for feature-based turbulence visualization that works on a compressed flow field representation. We use a wavelet-based compression scheme including run-length and entropy encoding, which can be decoded on the GPU and embedded into brick-based volume ray-casting. This enables a drastic reduction of the data to be streamed from disk to GPU memory. Our system derives turbulence properties directly from the velocity gradient tensor, and it either renders these properties in turn or generates and renders scalar feature volumes. The quality and efficiency of the system is demonstrated in the visualization of two unsteady turbulence simulations, each comprising a spatio-temporal resolution of 10244. On a desktop computer, the system can visualize each time step in 5 seconds, and it achieves about three times this rate for the visualization of a scalar feature volume.

Enhanced tumor therapy using vaccinia virus strain GLV-1h68 in combination with a ß-galactosidase-activatable prodrug seco-analog of duocarmycin SA.

Breast cancer is the most common cause of cancer-related death worldwide, thus remaining a crucial health problem among women despite advances in conventional therapy. Therefore, new alternative strategies are needed for effective diagnosis and treatment. One approach is the use of oncolytic viruses for gene-directed enzyme prodrug therapy. Here, the lacZ-carrying vaccinia virus (VACV) strain GLV-1h68 was used in combination with a ß-galactosidase-activatable prodrug derived from a seco-analog of the natural antibiotic duocarmycin SA. Tumor cell infection with the VACV strain GLV-1h68 led to production of ß-galactosidase, essential for the conversion of the prodrug to the toxic compound. Furthermore, drug-dependent cell kill and induction of the intrinsic apoptosis pathway in tumor cells was also observed on combination therapy using the prodrug and the GLV-1h68 strain, despite the fact that VACV strains encode antiapoptotic proteins. Moreover, GI-101A breast cancer xenografts were effectively treated by the combination therapy. In conclusion, the combination of a ß-galactosidase-activatable prodrug with a tumor-specific vaccinica virus strain encoding this enzyme, induced apoptosis in cultures of the human GI-101A breast cancer cells, in which a synergistic oncolytic effect was observed. Moreover, in vivo, additional prodrug treatment had beneficial effects on tumor regression in GLV-1h68-treated GI-101A-xenografted mice.

Use of an oncolytic vaccinia virus for the treatment of canine breast cancer in nude mice: preclinical development of a therapeutic agent.

Mammary cancers together with cancers of the skin account for about 60% of the total cancers occurring in dogs. The veterinary options for therapeutic management of canine mammary cancer are limited and prognosis for such patients is poor. In this study, we analyzed the functionality of the oncolytic vaccinia virus strain GLV-1h68 as a possible therapeutic agent for canine mammary cancer. Cell culture data demonstrated that GLV-1h68 efficiently infected and destroyed cells of the canine mammary adenoma cell line ZMTH3. Furthermore, after systemic administration this attenuated vaccinia virus strain primarily replicated in canine tumor xenografts in nude mice. The efficient tumor colonization process resulted in inhibition of tumor growth and drastic reduction of tumor size. This is the first report demonstrating that vaccinia virus is an effective tool for the therapy of canine mammary cancers, which might next be applied to dogs with breast tumors.

Renilla luciferase- Aequorea GFP (Ruc-GFP) fusion protein, a novel dual reporter for real-time imaging of gene expression in cell cultures and in live animals.

Light-emitting reporter proteins play an increasing role in the study of gene expression in vitro and in vivo. Here we present a ruc-gfp fusion gene construct generated by fusing a cDNA for Renilla luciferase (ruc) in-frame with a cDNA encoding the "humanized" GFP (gfp) from Aequorea. A plasmid containing the fusion gene construct was successfully transformed into, and expressed in, mammalian cells. The transformed cells exhibited both Renilla luciferase activity in the presence of coelenterazine and GFP fluorescence upon excitation with UV light. Spectrofluorometry of cells containing the Ruc-GFP fusion protein, in the absence of wavelengths capable of exciting GFP fluorescence but in the presence of the luciferase substrate, coelenterazine, showed an emission spectrum with two peaks at 475 nm and 508 nm. These two peaks correspond to the emission maximum of Renilla luciferase at 475 nm and that of GFP at 508 nm. The peak at 508 nm generated in the presence of coelenterazine alone (without UV excitation) is the result of intramolecular energy transfer from Renilla luciferase to Aequorea GFP. Southern analysis of genomic DNA purified from transformed Chinese hamster ovary (CHO) cells and fluorescence in situ hybridization (FISH) to metaphase chromosomes confirmed the integration of the ruc-gfp fusion gene on a single chromosome. The bifunctional Ruc-GFP fusion protein allows the detection of gene expression at the single-cell level based on green fluorescence, and in a group of cells based on luminescence emission. Furthermore, animal experiments revealed that light emission from the Ruc-GFP fusion protein can be detected externally in the organs or tissues of live animals bearing the gene construct.

A Renilla luciferase-Aequorea GFP (ruc-gfp) fusion gene construct permits real-time detection of promoter activation by exogenously administered mifepristone in vivo.

In this study, we used a steroid-induced promoter activation system as a molecular switch to study the exogenous activation of transgene expression. This promoter activation system consists of three components: (1) a steroidal inducer drug, mifepristone (RU486), which binds to (2) a chimeric transcription factor complex, consisting of the mutant human progesterone receptor fused to the yeast GAL4 DNA-binding domain and the activation domain of the herpes simplex virus protein VP16, and (3) a synthetic promoter, consisting of a series of GAL4 recognition sequences upstream of the adenovirus major late E1B TATA box, linked to a gene construct (ruc-gfp) encoding a Renilla luciferase- Aequorea green fluorescent protein (GFP) fusion protein. Transcription of the promoter-marker gene cassette is activated by the drug (mifepristone)-bound chimeric transcription factor complex. Monitoring of induced gene expression was carried out using a low-light video camera and a UV microscope to detect luciferase and GFP, respectively. Using this activation system, we observed a 10- to 25-fold activation, depending on the inducer dose, of both luciferase and GFP expression in transiently transfected cells in comparison to cells that were not exposed to mifepristone. We further demonstrated activation of gene expression from the promoter activation system in live animals. The plasmids PAP CMV-GL914VPc'SV, carrying the chimeric transcription factor cassette, and plasmid p17x4-TATA-ruc-gfp, carrying the ruc-gfp reporter gene construct, were co-injected into limb muscles of nude mice. Following DNA injection, mifepristone (50 micro g/kg) was delivered by intraperitoneal injection. Thirty-six hours after DNA and mifepristone injection, significant Renilla luciferase activity was detectable in the limb muscles. The promoter activation system was also demonstrated in limb muscles and livers of nude mice that had received transplants of ex vivo-modified cells, which were transiently transformed with both the chimeric activator plasmid and the ruc-gfp reporter plasmid prior to implantation. Significant Renilla activity and GFP fluorescence were detected externally in limb muscles and in the livers of anesthetized animals that had received an intraperitoneal injection of inducer. This external monitoring method for observing inducible gene expression in live animals will facilitate experimental studies of fundamental questions of biological and therapeutic relevance. It will be especially valuable for the analysis of gene function at specific stages of animal development. The method should also be of general use in gene therapy, since it permits simultaneous monitoring of the expression levels of light-emitting proteins and therapeutic proteins originating from the activation of identical promoters.

Microinjection and growth of bacteria in the cytosol of mammalian host cells.

Most facultative intracellular bacteria replicate in specialized phagosomes after being taken up by mammalian cells. Relatively few intracellular bacteria escape the phagosomal compartment with the help of cytolytic (pore-forming) proteins and replicate in the host cell cytosol. Without such toxins, intracellular bacteria cannot reach this cellular compartment. To circumvent the requirement of an "escape" step, we developed a procedure allowing the efficient direct injection of bacteria into the cytosol of mammalian cells. With this technique, we show that most bacteria, including extracellular bacteria and intracellular pathogens that normally reside in a vacuole, are unable to replicate in the cytosol of the mammalian cells. In contrast, microorganisms that replicate in the cytosol, such as Listeria monocytogenes, Shigella flexneri, and, to some extent, enteroinvasive Escherichia coli, are able to multiply in this cellular compartment after microinjection. Further L. monocytogenes with deletion in its PrfA-regulated hpt gene was found to be impaired in replication when injected into the cytosol. Complementation of the hpt mutation with a plasmid carrying the wild-type hpt gene restored the replication ability in the cytosol. These data indicate that cytosolic intracellular pathogens have evolved specific mechanisms to grow in this compartment of mammalian cells.

The p14ARF tumor suppressor protein facilitates nucleolar sequestration of hypoxia-inducible factor-1alpha (HIF-1alpha ) and inhibits HIF-1-mediated transcription.

Oncogenic alterations can influence tumor cell survival partly by affecting the activity of the hypoxia-inducible factor-1 (HIF-1) transcription factor. The alpha subunit of HIF-1 was found to be frequently overexpressed in advanced tumors, which was proposed to help the adaptation of tumor cells to hypoxia. Here we show that an important tumor suppressor protein, p14ARF (alternative reading frame product of the INK4A locus) can directly inhibit the transcriptional activity of HIF-1 by sequestering its alpha subunit into the nucleolus. The interaction requires neither p53 nor HDM2. This is one of the first reports that describe the interaction of p14ARF with a protein besides HDM2, which may define a p53-independent tumor suppressor activity for p14ARF.

A study of protein-protein interactions in living cells using luminescence resonance energy transfer (LRET) from Renilla luciferase to Aequorea GFP.

We have previously reported that Escherichia coli and mammalian cells containing a fusion protein consisting of the Renilla luciferase linked to Aequorea GFP exhibited luminescence resonance energy transfer (LRET) from luciferase to GFP in the presence of coelenterazine. In this paper, we describe the construction of two gene fusions in which the cDNA for insulin-like growth factor II (IGF-II) is connected to the cDNA for a "humanized" GFP, and the cDNA for insulin-like growth factor binding protein 6 (IGFBP-6) is linked to a cDNA encoding the Renilla luciferase (RUC). The expression of the fusion gene constructs in CHO cells resulted in single polypeptides with the molecular weights expected for IGF-II-GFP and IGFBP-6-RUC, respectively, based on the use of antibodies against GFP and Renilla luciferase. The secretion of IGF-II-GFP from CHO cells was verified by fluorescence microscopy and the presence of IGFBP-6-RUC in the culture medium was confirmed by luminometry. The interaction between the two known binding partners, IGF-II and IGFBP-6, was monitored by measuring LRET from the IGFBP-6-RUC protein to IGF-II-GFP in the presence of coelenterazine, using a low-light imaging system and spectrofluorometry. Based on these data, luciferase-to-GFP LRET holds great promise for the study of protein-protein interactions in eukaryotic cells in real time.

Detection of GDNF secretion in glial cell culture and from transformed cell implants in the brains of live animals.

Current ex vivo gene therapy for Parkinson's disease using glial cell line-derived neurotrophic factor (GDNF) is limited by the lack of a monitoring mechanism to determine the expression of GDNF once the cells or other vehicles are transferred into animal models. The purpose of this study was to test whether a Renilla luciferase (RUC)-GDNF fusion protein secreted by the genetically engineered glial cell line RG-1 could be measured photometrically in cerebrospinal fluid (CSF). RG-1 was constructed by permanent transformation with a plasmid DNA construct that contains a GDNF cDNA (gdnf) fused to a RUC cDNA (ruc). The fusion protein secreted by RG-1 was shown to retain both GDNF and RUC activity. The concentration of GDNF determined by enzyme-linked immunoadsorbent assay (ELISA) was correlated with the light emission detected by assaying for RUC bioluminescence in RG-1 culture medium, indicating that RUC can be used as a reporter for GDNF in vitro. The cells were then implanted into rat brain (n=20), and the cisternal CSF was analyzed. Bioluminescence was successfully detected in the CSF samples, and was quantified over a period of 25 days, while Western blotting and ELISA failed to detect GDNF in CSF, presumably because the concentration of the RUC-GDNF fusion was too low. This study demonstrates that the transformed glial cell line RG-1 offers a sensitive self-reporting assay for GDNF expression.

Endocrine status in elderly men with lower urinary tract symptoms: correlation of age, hormonal status, and lower urinary tract function. The Prostate Study Group of the Austrian Society of Urology.

OBJECTIVES: To correlate endocrine parameters in elderly men with lower urinary tract symptoms (LUTS) to patient age and clinical parameters such as prostate volume, prostate-specific antigen (PSA) levels, and uroflowmetry and to compare the clinical and endocrinologic parameters in men with or without hypogonadism. METHODS: Men (40 years old or older) with untreated LUTS as defined by an International Prostate Symptom Score (IPSS) of 7 or greater due to benign prostatic hyperplasia were included in this study and underwent the following investigations: IPSS, free uroflow study, postvoid residual volume, transrectal ultrasound for assessment of prostate volume, serum PSA determination, and an endocrine study, including testosterone, human luteinizing hormone, human follicle-stimulating hormone, prolactin, dehydroepiandrostendione-sulphate (DHEA-S), and prolactin. RESULTS: Three hundred twelve men (mean age 62.8 +/- 10.6 years, range 40 to 91) were analyzed. The serum levels of estradiol (correlation coefficient [r] = 0.19), human luteinizing hormone (r = 0.32), human follicle-stimulating hormone (r = 0.19), and DHEA-S (r = -0.39) correlated (P <0.05) with age; no such correlation was seen for testosterone (r = 0.04; P0.05) or prolactin (r = 0.09; P0.05). Estradiol (but not testosterone) correlated (r = 0.17, P = 0.01) with prostate volume. The peak flow rate and PSA did not correlate with any endocrinologic parameter. Hypogonadism (serum testosterone less than 3.0 ng/mL) was detected in 22.1% of patients and had no impact on clinical (IPSS, peak flow rate, prostate volume, and PSA level) or endocrine (human luteinizing hormone, human follicle-stimulating hormone, estradiol, prolactin, and DHEA-S) parameters. CONCLUSIONS: A number of age-related endocrine changes are seen in elderly men with LUTS. Hypogonadism is seen in approximately one fifth of elderly men with LUTS, but in our study it had no impact on symptom status, PSA level, prostate volume, uroflowmetry, or endocrine parameters.

Visualizing and quantifying protein secretion using a Renilla luciferase-GFP fusion protein.

We have shown previously that an engineered form of Renilla luciferase (SRUC) can be secreted as a functional enzyme by mammalian cells, and that fusing wild-type Renilla luciferase with the green fluorescent protein from Aequorea victoria (GFP) yields a chimeric protein retaining light-emission properties similar to that of unfused Renilla luciferase and GFP. In the work presented here, SRUC was fused with GFP to determine whether it could be used to both visualize and quantify protein secretion in mammalian cells. Simian COS-7 and Chinese hamster ovary (CHO) cells were transiently transfected with gene constructs encoding a secreted or an intracellular version of a Renilla luciferase-GFP fusion protein. Renilla luciferase activity was measured from COS-7 cell lysates and culture media, and GFP activity was detected in CHO cells using fluorescence microscopy. Data indicated that the SRUC-GFP fusion protein was secreted as a chimeric protein that had both Renilla luciferase and GFP activity. This fusion protein could be a useful marker for the study of protein secretion in mammalian cells.

Mer22-related sequence elements form pericentric repetitive DNA families in primates.

We describe a novel repetitive DNA element isolated from three primate species belonging to the family Cercopithecidae. The unusually long 2.6-kb repeat unit of this DNA element is present in high copy number in the pericentromeric region of one pair of chromosomes in both baboon and macaque, forming chromosome-specific satellite-like DNA families. Besides these two very closely related species, the novel DNA element was also detected in the more distantly related African green monkey. However, the copy number of the repeat unit in this species is significantly lower than in macaque and baboon. Sequence analysis revealed that the repeat units of the new repetitive element show similarity to the human MER22 repeat and the Y chromosome-specific TTY2 element, which also exhibits retroelement-like features. Database searches indicate that tandemly arranged MER22-related DNA sequences can also be found in human, raising the possibility that these DNA elements may correspond to a novel primate-specific repetitive DNA group. Recent studies indicate that chromosome-specific pericentric repetitive elements, besides their potential involvement in centromere function, also facilitate homolog recognition during meiosis. In addition, rapid expansion of retroelements in the pericentric regions of chromosomes during interspecific hybridization has been described. In light of these data, we hypothesize that the novel repetitive element described here might have been involved in the speciation of the family Cercopithecidae.

Genotyping the baboon ABO histo-blood group locus by two-color fluorescence SSCP.

The use of baboons in experimental medicine, especially as organ and tissue donors, would be facilitated by the availability of ABO histo-blood group O animals, which are currently rare. To meet our need in breeding and identifying such animals, we have developed a single-stranded conformational polymorphism (SSCP)-based genotyping assay using fluorescence detection. Various buffers and labeling schemes were evaluated to optimize allele discrimination. Through the use of a nested PCR protocol, single-cell sensitivity was achieved, making the assay applicable to preimplantation diagnosis following in vitro fertilization. We discuss the comparative advantages of SSCP vs. alternative methodologies for genotyping.

An alternative intronic promoter of the Bombyx A3 cytoplasmic actin gene exhibits a high level of transcriptional activity in mammalian cells.

Previous observations have indicated that the Bombyx mori gene for A3 cytoplasmic actin and vertebrate actin genes might make use of similar mechanisms for regulation of gene expression. To examine the suggested similarities, we have analyzed the expression of a LacZ reporter plasmid construct containing the 5' and 3' regulatory regions and the first intron of the A3 actin gene in a variety of vertebrate cell lines. We found that this silkworm expression cassette could drive expression of foreign genes in both mammalian and avian cell lines. Detailed analysis, however, indicated that neither the CArG box nor any of the promoter elements previously identified in the A3 actin gene were required for expression in mammalian cells. On the other hand, the first intron contained an efficient promoter, exhibiting in mouse cells a transcriptional activity comparable to that of the SV40 early promoter. The first intron of the A3 gene was also found to contain enhancer-like DNA elements that could stimulate the heterologous SV40 early promoter in mammalian cells. Promoter activity of the first intron of the A3 actin gene has not been observed previously. Recently however, we described a rare A3 actin mRNA isoform in B. mori cells, which initiates within the first intron. We suggest that the identified intronic promoter may be active not only in vertebrate cells but also in silkworm, and that it regulates the synthesis of the alternative A3 actin mRNA isoform. The characteristics of the 5' regulatory region of the A3 gene described here can also be exploited in the construction of bi-functional insect-mammalian expression vectors.

Molecular characterization of a stably transformed Bombyx mori cell line: identification of alternative transcriptional initiation sites of the A3 cytoplasmic actin gene.

We have isolated a stably transformed Bormbyx mori cell line containing a novel selectable marker gene, puromycin N-acetyl transferase, under control of transcriptional regulatory signals from the A3 cytoplasmic actin gene. By using this cell line we have identified alternative transcriptional initiation sites for the A3 actin gene. One of these start sites is located approximately 35 bp upstream from the previously determined transcription initiation site. The two mRNA start sites are utilized with a similar efficiencies in the BmN cell line. In addition, we detected transcripts that initiated in the first intron of the A3 actin gene. These transcripts may be synthesised under control of an alternative promoter. The stably transformed B. mori cell line used in this study was also extensively characterized. Integration of the plasmid molecules into the host genome was demonstrated by Southern and in situ hybridization analyses. Establishment and characterization of stably transformed insect cell lines, like the one described here, represents an important step in the development of nonlytic insect expression systems.