Biomedical subjects

Holger Kalthoff

Publications and source records attributed to Holger Kalthoff.

45 records · Page 3Linked to original sources

PubMed2002

Wortmannin inhibits growth of human non-small-cell lung cancer in vitro and in vivo.

BACKGROUND AND AIMS: Recently we demonstrated that phosphatidylinositol 3-kinase (PI3K) is overexpressed in human lung cancer. This study evaluated whether the PI3K inhibiting agent wortmannin affects proliferation of human lung cancer cells in vitro and in vivo. METHODS: Effects of exposure of human non-small-cell lung cancer (NSCLC) cells (KNS-62, Colo-699) to wortmannin were investigated in vitro by proliferation, cytotoxicity, and DNA fragmentation assays. In vivo we examined the effects of blocking PI3K by wortmannin prior to xenotransplantation of human NSCLC cells into SCID-bg mice and the effect of systemic wortmannin administration following intrapulmonary xenotransplantation of human NSCLC. RESULTS: Exposure of KNS-62 and Colo-699 lung cancer cells to wortmannin inhibited proliferation in correlation to concentration in vitro. In vivo the blocking of PI3K by wortmannin prior to xenotransplantation caused a significant delay in the growth of subcutaneously induced tumors. Systemic wortmannin administration increased mean survival after intrapulmonary xenotransplantation of human NSCLC significantly by 38% and 47%. CONCLUSIONS: These data suggest inhibition of PI3K activity as a potential target for treatment of human NSCLC. Systemic toxicity of wortmannin requires development of improved PI3K inhibitors with favorable pharmacological properties.

Androstadienes↗

PubMed2002

Smad4/DPC4-dependent regulation of biglycan gene expression by transforming growth factor-beta in pancreatic tumor cells.

Overexpression of the small leucine-rich proteoglycan biglycan (BGN) in fibrosis and desmoplasia results from enhanced activity of transforming growth factor-beta (TGF-beta). In pancreatic adenocarcinoma, the tumor cells themselves may contribute to BGN synthesis in vivo, since 8 of 18 different pancreatic carcinoma cell lines constitutively expressed BGN mRNA, as shown by reverse transcription-PCR analysis. In PANC-1 cells, TGF-beta1 dramatically stimulated BGN mRNA accumulation through a BGN transcription-independent, cycloheximide-sensitive mechanism and strongly increased the synthesis and release of the proteoglycan form of BGN. The ability of TGF-beta1 to induce BGN mRNA was critically dependent on Smad signaling, since 1) the up-regulation of BGN mRNA was preceded by a marked increase in Smad2 phosphorylation in TGF-beta1-treated PANC-1 cells, 2) TGF-beta1 was unable to induce BGN mRNA in pancreatic carcinoma cell lines that carry homozygous deletions of the Smad4/DPC4 gene, 3) inhibition of the Smad pathway in PANC-1 cells by transfection with a dominant negative Smad4/DPC4 mutant significantly reduced TGF-beta1-induced BGN mRNA expression, 4) stable reintroduction of wild type Smad4/DPC4 into Smad4-null CFPAC-1 cells restored the TGF-beta1 effect, and 5) overexpression of Smad2 and Smad3 in PANC-1 cells augmented TGF-beta1 induction of BGN mRNA, whereas forced expression of Smad7, an inhibitory Smad, effectively blocked it. These results clearly show that a functional Smad pathway is crucial for TGF-beta regulation of BGN mRNA expression. Since BGN has been shown to inhibit growth of pancreatic cancer cells, the Smad4/DPC4 mediation of the TGF-beta effect may represent a novel tumor suppressor function for Smad4/DPC4: antiproliferation via expression of autoinhibitory BGN.

Biglycan↗

PubMed2002

Dual regulation of telomerase activity through c-Myc-dependent inhibition and alternative splicing of hTERT.

Telomerase is believed to be induced upon proliferation and inhibited when cells differentiate. Thus, regulation of telomerase activity could be an important mechanism to limit growth of normal and cancer cells. Using transforming growth factor-beta 1 (TGF-beta1), which is known to control proliferation in epithelial cells, we now demonstrate that in the human HaCaT skin keratinocytes, TGF-beta1 downregulated c-Myc, and this blocked proliferation. This also caused a decrease in hTERT expression, which in turn inhibited telomerase activity. Overexpressing hTERT recovered telomerase activity but not proliferation, whereas constitutive expression of c-Myc recovered proliferation and hTERT expression. Nevertheless, telomerase remained inhibited, thus dissociating proliferation and telomerase activity. In addition, we show that TGF-beta1 inhibited telomerase activity despite ongoing hTERT transcription by inducing loss of the full-length hTERT transcript (mediating telomerase activity) and retaining high expression of the inactive beta variant. These changes in the splicing pattern reversed upon TGF-beta1 removal, as did inhibition of telomerase activity, suggesting that alternative splicing may represent a novel mechanism of telomerase regulation by TGF-beta1. In addition, we show that destruction of tissue integrity (in a model for epidermal blistering) resulted in a rapid induction of the inactive beta variant, whereas tissue regeneration (formation of a stratified epithelium) correlated with a shift to the active full-length transcript, which is the dominant form in intact epidermis. Thus alternative splicing may not be restricted to TGF-beta1 but may add a more general mechanism of hTERT regulation in epidermal cells.

Alternative Splicing↗

PubMed2002

Ganciclovir prodrug therapy is effective in a murine xenotransplant model of human lung cancer.

BACKGROUND: Therapy failures have been reported for retroviral gene transfer of herpes simplex virus thymidine kinase (HSV-TK) gene followed by systemic ganciclovir application in human lung cancer. Use of the HSV-TK mutant TK30 in combination with a VSV-G pseudotyped retroviral vector was found to enhance the efficacy of prodrug therapy. The present study evaluated this therapeutic strategy in human non-small cell lung cancer cell lines in a preclinical murine xenotransplant model. METHODS: Intrathoracally induced by HSV-TK30 transduced non-small cell lung cancer cell lines Colo 699 (adenocarcinoma) and KNS 62 (squamous cell carcinoma) were monitored for local tumor growth, survival, and metastases. So-called bystander effects were investigated in tumors consisting of as little as 25% TK30 transfected cells and by analysis of gap junctional protein connexin-43 expression. RESULTS: Survival was significantly prolonged, and tumor growth and pleural metastases were reduced in HSV-TK30-positive tumors of both cell lines. A significant therapeutic effect in bystander experiments was observed in squamous cell carcinoma. This was correlated with higher expression of connexin-43. CONCLUSIONS: Delivery of HSV-TK30 in a VSV-G pseudotyped retroviral vector and subsequent ganciclovir application provided therapeutic efficacy. Despite of low transduction rates achievable in gene transfer in situ, prodrug therapy appears to be feasible in tumor cells with efficient bystander effects.

Adenocarcinoma↗

PubMed2002

Detection and prognostic impact of disseminated tumor cells in pancreatic carcinoma.

BACKGROUND/AIMS: Metastatic disease determines the prognosis of patients with pancreatic cancer. Current routine staging methods often underestimate the tumor stage because they do not include the search for disseminated tumor cells that spread early in different compartments of the body. Immunohistochemical and molecular methods developed recently are able to detect these cells in multiple compartments of the body. METHODS: The current status of the detection and the prognostic impact of disseminated tumor cells detected in lymph nodes, bone marrow, blood and peritoneal lavage of patients with pancreatic carcinoma are reviewed. RESULTS: Disseminated tumor cells can be detected in different compartments of the body even in early tumor stages and when a resection of the primary tumor in curative intention was performed. Furthermore, the detection of these cells has importance for the prognosis and therefore will have therapeutic implications. Standardization of the methods is a prerequisite for further studies. CONCLUSION: The detection of disseminated tumor cells should be included into studies to reveal that this increased staging has an prognostic impact and can be useful for therapeutic decisions in patients with pancreatic carcinoma.

Bone Marrow↗

PubMed2002

Characterization of the sialate-7(9)-O-acetyltransferase from the microsomes of human colonic mucosa.

Sialic acids present on human colonic mucins are highly O-acetylated, however, little is known about the underlying enzymatic activity required for O-acetylation in this tissue. Here we report on the substrate specificity, subcellular localization and characterization of the sialate-7(9)-O-acetyltransferase in normal human colonic mucosa. Using CMP-Neu5Ac, the most efficient acceptor substrate of all those tested, the enzymatic activity was found to be optimal at 37 degrees C, with a pH optimum of 7.0. Activity was also found to be dependent on protein, CMP-Neu5Ac (Km: 59.2 microM) and AcCoA (Km: 6.1 microM) concentrations, as well as membrane integrity. The enzyme's activity could be inhibited by CoA with a Ki of 11.9 microM. In addition, enzymatic activity was found to be localized in the Golgi-enriched membrane fraction. The nature of the O-acetylated products formed were verified with the aid of chromatographic and enzymatic techniques. The main product was 9-O-acetylated Neu5Ac, with a significant amount of oligo-O-acetylated Neu5Ac also being detected. The utilization of CMP-Neu5Ac as the acceptor substrate was confirmed by the isolation and characterization of the putative product, CMP-Neu5,9Ac2, using ion-exchange chromatography. The ability of CMP-Neu5,9Ac2 to act as a sialic acid donor for sialyltransferases represents the conclusive demonstration for the formation of CMP-Neu5,9Ac2.

Acetylation↗

PubMed2002

[Comparative analysis of metastatic variants from the colorectal tumor cell line HT-29]

OBJECTIVE: To analyze the relationship between metastatic potential and related facters of colorectal tumor cell lines. METHODS: The variants HT-29c and HT-29d cell lines derived from the selection of HT-29 cells were injected into nude rats and the metastatic potential of the two tumor cell variants was analyzed. Expression of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) were measured with ELISA in vitro in colorectal carcinoma cell lines WiDr, HT-29 and HT-29d. Expression of carcinoembryonic antigen (CEA) and phosphoinositide 3-kinase (PI3-Kinase) were determined with immunohistochemistry, (IHC) in vitro and in vivo in WiDr, HT-29 and HT-29d cell lines. In addition, CEA expression was demonstrated with fluorescence activated cell sorter (FACS) in vitro. RESULTS: The liver metastasis rate of the variant HT-29d (with 4 cycles of selection), increased as compared with that of parental HT-29 cells and that of variant HT-29b cells (with 2 cycle of selection). The uPA concentration of variant HT-29d cell line was significantly higher than that of the non metastatic WiDr and the low metastatic HT-29 cells (P<0.05). The variant HT-29d cells produced stronger PI3-kinase expression as compared with the non-anetastatic WiDr cells and the low metastatic HT-29 cells in vivo. CONCLUSION: The selected variant cell lines can exhibit an enhanced metastatic potential. The level of uPA and PAI-1 are positively correlated with the metastatic capacity of tumor cells. The expression of PI3 kinasecorrelates with tumor development and metastasis.

Journal Article↗

PubMedDate not supplied

Gene expression profiling of microdissected pancreatic ductal carcinomas using high-density DNA microarrays.

Pancreatic ductal adenocarcinoma (PDAC) remains an important cause of malignancy-related death and is the eighth most common cancer with the lowest overall 5-year relative survival rate. To identify new molecular markers and candidates for new therapeutic regimens, we investigated the gene expression profile of microdissected cells from 11 normal pancreatic ducts, 14 samples of PDAC, and 4 well-characterized pancreatic cancer cell lines using the Affymetrix U133 GeneChip set. RNA was extracted from microdissected samples and cell lines, amplified, and labeled using a repetitive in vitro transcription protocol. Differentially expressed genes were identified using the significance analysis of microarrays program. We found 616 differentially expressed genes. Within these, 140 were also identified in PDAC by others, such as Galectin-1, Galectin-3, and MT-SP2. We validated the differential expression of several genes (e.g., CENPF, MCM2, MCM7, RAMP, IRAK1, and PTTG1) in PDAC by immunohistochemistry and reverse transcription polymerase chain reaction. We present a whole genome expression study of microdissected tissues from PDAC, from microdissected normal ductal pancreatic cells and pancreatic cancer cell lines using high-density microarrays. Within the panel of genes, we identified novel differentially expressed genes, which have not been associated with the pathogenesis of PDAC before.

Aged↗

PubMedDate not supplied

Resistance developing after long-term ganciclovir prodrug treatment in a preclinical model of NSCLC.

BACKGROUND: We recently demonstrated a 100% increase in the survival period with ganciclovir (GCV) therapy in mice hearing orthotopic HSV-TK-positive non-small cell lung cancer (NSCLC) tumors. However, long-term survival was not achieved. The aim of the present study was to evaluate tumor growth during extended GCV therapy and to monitor the herpes simplex virus thymidine kinase (HSV-TK) gene and protein in tumors at different time points. MATERIALS AND METHODS: The human NSCLC cell line KNS 62 was retrovirally transduced with the HSV-TK30 gene. Cell suspensions in which 100% or 25% of the cells were TK30-positive were inoculated subcutaneously in SCID bg mice. Tumor growth was evaluated during GCV therapy and HSV-TK DNA, RNA and protein were analyzed at different time points using PCR, RT-PCR and immunoblotting. RESULTS: HSV-TK DNA, RNA and TK30 protein were demonstrated in the tumors 21 days after subcutaneous tumor inoculation. TK-positive tumors regressed during GCV therapy and tumors in which 25% of the cells were TK-positive grew significantly more slowly than control tumors did. After 4 weeks of GCV therapy, HSV-TK DNA, RNA and TK protein were not detectable in the remaining tumors, which were therefore resistant to further GCV therapy. CONCLUSION: Prodrug therapy of the NSCLC cell line KNS 62, including bystander effects, is sufficient. Nevertheless, GCV-resistant tumors develop after functional loss of the TK gene. In the clinical context, further studies will need to evaluate immunological bystander effects or combinations with other drugs.

Animals↗