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Biomedical subjects

I R Hart

Publications and source records attributed to I R Hart.

At least 73 records · Page 4Linked to original sources
PubMed1994

Experimental metastasis and differentiation of murine melanoma cells: actions and interactions of factors affecting different intracellular signalling pathways.

Various factors that modulate the differentiation of malignant cells are known to affect their experimental metastatic potential (EMP), or lung colonization after intravenous injection into syngeneic animals. However, some results and conclusions on the relation between cell differentiation and metastasis have appeared to conflict. We have reanalysed this by measurement of EMP of B16 melanoma sublines after culture with agents or conditions that acted on differentiation through various intracellular pathways. All tested agents did affect the EMP. EMP was usually positively correlated with differentiation under diverse conditions, but exceptions showed that there is no direct causal connection. Nor could all findings be explained in terms of cell proliferation or expression of major histocompatibility antigens. Some data helped to explain disparities between previous reports. Specific novel findings included the following. The stimulation of EMP by melanocyte-stimulating hormone (MSH) as well as all other tested effects of MSH were prevented by extended exposure to 12-O-tetradecanoyl phorbol acetate (TPA), suggesting a requirement for protein kinase C activity as well as G-protein coupling in MSH action. Cells grown with cholera toxin were always more differentiated than untreated cells, but the EMP could be either markedly increased or markedly decreased by cholera toxin under different conditions. The basic culture medium apparently determined this striking reversal. The EMP was also significantly affected by the extracellular pH.

Animals↗
PubMed1994

Proliferating cell nuclear antigen expression in non-cycling cells may be induced by growth factors in vivo.

The proliferating cell nuclear antigen (PCNA) is required for DNA replication and DNA nucleotide excision repair. Considerable evidence points to PCNA expression being a marker of proliferation in many situations. However, while levels of PCNA are normally very low in non-cycling tissues, high levels of the protein have been observed in the normal tissues surrounding human breast and pancreatic tumours. Using two model systems we have shown that PCNA is induced in non-cycling cells by adjacent transplanted tumour cells and that this phenomenon may be mimicked by the in vivo administration of growth factors (transforming growth factor alpha and epidermal growth factor). These data suggest that tumours may elaborate factors that induce PCNA expression in nearby normal cells. PCNA induction the normal cells surrounding tumours is a direct example of the effect of tumour cells on normal surrounding tissues. This effect may prove to be a useful parameter in the analysis of tumour-host interactions.

Animals↗
PubMed1994

Targeting of cytokine gene expression to malignant melanoma cells using tissue specific promoter sequences.

BACKGROUND: Transduction of tumor cells in vitro with cDNAs encoding various cytokines and/or immune accessory molecules has been shown to diminish or eliminate tumorigenicity when such cells are returned in vivo to syngeneic animals. One method being explored for in situ gene therapy is to use tissue-specific promoters to direct expression of the therapeutic genes to the tumour cells. DESIGN: This study used the 5' flanking region of the murine tyrosinase gene to direct expression of three different cytokine genes [murine interleukin 2 (IL-2), IL-4 and macrophage colony-stimulating factor (M-CSF)] specifically to murine melanoma cells. RESULTS: Expression of the IL-2 gene, from 2.5 kbp of the 5' flanking region of the murine tyrosinase gene, was detected in 11 out of 55 puromycin-resistant B16 clones isolated after transfection. The highest producing clone secreted 2000 pg/ml/10(6) cells/48 hours as determined by enzyme-linked immunosorbent assay. The IL-2 was tested for biological activity by its ability to stimulate proliferation of the IL-2 dependent CTLL cell line. No detectable level of IL-2 expression occurred in 58 clones of drug-resistant NIH 3T3 cells derived after transfection with the same construct. Similar results were obtained following transfection of these two cell lines with the tyrosinase-IL-4 minigene construct. Expression of IL-2 in the murine melanoma cells completely abrogated their tumorigenicity in syngeneic mice. However, progressively growing tumours were produced from clones in which the IL-2 gene was no longer expressed (as determined by reverse transcriptase polymerase chain reaction). Direct injection of DNA encoding cytokine genes, expressed from the tyrosinase promoter, into established B16 melanomas in syngeneic mice resulted in gene expression within the tumour mass. While no change in tumour growth was observed following such treatment, the results demonstrate that direct injection of naked DNA into a neoplasm can result in uptake and expression of cytokine genes up to 16 days post-injection. CONCLUSION: The use of tissue-specific promoters can limit expression to the required target cell, while the choice of appropriate gene should result in an alteration in tumour burden.

Animals↗
PubMed1994

Targeted therapy for malignant melanoma.

Malignant melanoma is a tumor that offers unique possibilities for approaches to targeted therapy by virtue of the pigment biosynthesis pathway. Such approaches may seek to use the incorporation of toxic intermediates or to use the natural transcriptional control of genes coding for enzymes involved in melanin formation to regulate gene therapy. In this paper, we describe how the 5'-flanking sequences of the genes for tyrosinase or tyrosinase-related protein 1 can be used to drive expression of complementary DNA, coding for immunity-stimulating proteins or for drug-activating enzymes, specifically in melanoma cells. The combination of the tissue specificity resulting from these techniques, coupled with innovative delivery systems, should provide the maximum available therapeutic index and lead to the design of new treatments with limited side effects.

Gene Transfer Techniques↗
PubMed1993

Growth of melanocytic cells is associated with down-regulation of protein kinase C alpha, delta, and epsilon isoforms. Possible role of diacylglycerol.

Protein kinase C (PKC) down-regulation has been shown to correlate with the growth of murine melanocytic cells in culture (Brooks, G., Wilson, R. E., Dooley, T. P., Goss, M. W., and Hart, I. R. (1991) Cancer Res. 51, 3281-3288). We now show that PKC alpha, delta, epsilon, and zeta isoforms are present at the protein level in quiescent, non-transformed Mel-ab melanocytes, maintained in the absence of phorbol ester. Proliferation of Mel-ab cells, achieved by incubation in the continual presence of phorbol 12,13-dibutyrate, was associated with a down-regulation of the PKC alpha, delta, and epsilon isozymes. Examination of two transformed syngeneic lines (the B16 murine melanoma and the long terminal repeat Ras.2 line), that grew in the absence of exogenous phorbol esters, showed that PKC alpha protein levels were either partially down-regulated or unaffected, the PKC delta and epsilon isoforms were down-regulated completely, and the levels of PKC zeta protein remained unaltered relative to quiescent Mel-ab cells. Basal levels of total diacylglycerol were elevated 5-fold in B16 melanoma cells compared with levels found in quiescent or proliferating Mel-ab melanocytes and appear to arise largely from the breakdown of phosphatidylinositol phospholipids accompanied by a significant rise in phospholipase C activity. Hourly treatments of quiescent Mel-ab melanocytes with the synthetic diacylglycerol analogue, 1,2-dioctanoyl-sn-glycerol, for 24 h, resulted in an induction of DNA synthesis which was associated with a significant down-regulation of PKC levels mediated largely via post-translational rather than transcriptional mechanisms. These results show for the first time that specific isoforms of PKC are down-regulated at the protein level during proliferation of murine melanocytic cells and suggest that the constitutive down-regulation of PKC in transformed melanoma cells may arise as a consequence of elevated endogenous phosphatidylinositol-derived diacylglycerol levels.

Animals↗
PubMed1993

Use of tissue-specific expression of the herpes simplex virus thymidine kinase gene to inhibit growth of established murine melanomas following direct intratumoral injection of DNA.

We report here the use of the 5' flanking region of the murine tyrosinase gene to direct expression of the herpes simplex virus thymidine kinase (tk) gene specifically to murine melanoma cells, whilst not permitting expression in a range of other cell types. Expression of the herpes simplex virus tk gene from the tyrosinase promoter in melanoma cells rendered them sensitive to killing by ganciclovir (100% cell death of a tk-expressing B16 clone after 12 days in culture at 1 microgram/ml ganciclovir). We also observed a substantial bystander killing effect when expressing cells were mixed with nontransfected parental B16 cells. When transfected murine melanoma cells expressing tk were injected into syngeneic mice both their tumorigenicity and experimental metastatic potential were abrogated completely when the mice were treated with ganciclovir (27 of 28 mice treated with water developed progressively growing tumors versus 1 of 30 in the ganciclovir-treated group). Direct injection of the tk gene under control of the tyrosinase promoter into established tumors in mice, followed by treatment with ganciclovir, led to significant reductions in resultant tumor size relative to the size of tumor developing in mice treated with water (median tumor weight, 1.65 g versus 2.75 g). Therefore, direct transfer of recombinant genes by injection of DNA can significantly reduce established tumor burden in vivo.

Animals↗
PubMed1993

In vitro and in vivo targeting of gene expression to melanoma cells.

Gene therapy protocols for cancer usually involve removal of tumor cells, culture in vitro to allow gene transfer, and subsequent reintroduction in vivo. Targeting therapeutic genes to tumor cells in situ requires an accuracy of gene delivery that currently is not possible with the use of existing techniques. To overcome these limitations we have used two promoters, which are preferentially active in melanocytic cells, to direct gene expression specifically to melanoma cells both in vitro and in vivo. Here we describe experiments showing that as little as 769 base pairs of the 5'-flanking regions of the tyrosinase, and 1.4 kilobase pair of the tyrosinase-related protein 1, genes are sufficient to direct expression of the beta-galactosidase gene to both human and murine melanoma cells and melanocytes, while not permitting expression in a range of other cell types in vitro. These promoters showed high levels of activity in 12 of 14 murine and human melanoma cell lines tested but showed only basal levels of activity, similar to that of a promoterless construct, in a range of 12 other cell types. Cell type specificity is maintained when the construct is delivered to cells either by physical means or by inclusion of the cell type-specific expression cassette into a retroviral vector. Direct injection of DNA, encoding the beta-galactosidase gene expressed from either promoter, into established B16 melanomas or Colo 26 tumors in syngeneic mice resulted in extensive transduction of tumor cells in the B16 melanomas (approximately 10% of tumor cells expressing 10 days after DNA injection), whereas no blue-staining cells were seen in the Colo 26 tumors. The reporter gene was expressed in melanoma cells and in some normal melanocytes but not in other surrounding normal tissue. We propose that the combination of a tissue-specific promoter driving a therapeutic gene, with delivery of such a construct directly to sites of tumor growth in vivo, either by direct DNA injection or by retroviral infection, may provide significantly enhanced safety for gene therapy for solid tumors.

Animals↗
PubMed1993

Expression and function of the CD44 glycoprotein in melanoma cell lines.

We have analysed a panel of murine and human melanoma cell lines for expression of the glycoprotein CD44. All 12 cell lines examined expressed CD44 at their cell surfaces, as demonstrated by fluorocytometric analysis using monoclonal antibodies (MAbs) IM7 and F10.44.2. Northern analysis revealed three transcript sizes that were 4.5, 2.2, and 1.5 kb in the human cell lines and 4.5, 3.0, and 1.5 kb in the murine cell lines. Levels of mRNA did not correlate with level of surface expression, which was highly variable between the cell lines. RT-PCR analysis of mRNA revealed that the major band identified was the expected 792 bp fragment indicative of the CD44H haemopoietic form, compared to a 1194 bp form found in the human colorectal adenocarcinoma cell line HT29, indicative of the CD44E epithelial form. There was no evidence of variant CD44 mRNA in our panel of melanomas. Functional assays revealed no clear correlation between the level of cell surface CD44 and the ability of the melanoma cell lines to adhere to hyaluronate. Rather adherence appeared to relate to the activation status of CD44 on the different cell lines as a consequence of MAb stimulation (e.g. the 1735P line demonstrated a 46.2 +/- 5.7% adherence in an inactivated state versus 62.4 +/- 5.6% adherence in an activated state to 5 mg/ml hyaluronate) and suggests that the functional capacity of CD44 expressed by melanoma cells may be modified more by activation state than by RNA splicing.

Adenocarcinoma↗
PubMed1993

Mechanisms underlying invasion and metastasis.

Despite continuing advances in the molecular characterization of the events promoting metastasis, no significant impact has been made on therapy or survival rates for patients with advanced tumors. Over the past 12 months there has been a general consolidation of the emerging evidence indicating that tumor spread is regulated by mechanisms involving disturbances in normal homeostatic processes. This review discusses these developments and also highlights the active role of peritumoral stroma in modulating invasion and spread of cancer.

Cell Adhesion↗
PubMed1993

E-cadherin expression in colorectal cancer. An immunocytochemical and in situ hybridization study.

Expression of the epithelial-specific adhesion molecule E-cadherin has been assessed in paraffin-embedded tissue from a series of 72 colorectal carcinomas. Using immunocytochemistry and in situ hybridization it was found that E-cadherin expression was related inversely to tumor differentiation. Out of 44 well- and moderately differentiated tumors, 36 expressed good positivity, whereas 24 of 28 poorly differentiated tumors were E-cadherin-negative. Classification by Dukes stage revealed a highly significant difference (P << 0.001) between A and B (32 positive, four negative) and C1 and C2 (seven positive, 29 negative) stages in terms of immunoreactivity. Of the 32 lymph node metastases studied, 20 were negative for E-cadherin expression, as were seven of eight liver metastases. These results indicate that the down-regulation of E-cadherin levels in vivo is associated with the dedifferentiation, progression, and metastasis of colorectal cancer.

Adenocarcinoma↗
PubMed1992

Differential induction of 12-O-tetradecanoylphorbol-13-acetate sequence gene expression in murine melanocytes and melanoma cells.

We previously showed that growth of the nontumorigenic, immortal murine melanocyte line Mel-ab correlates with the depletion of protein kinase C (PKC), whereas quiescence is associated with elevated levels of this enzyme (Brooks G, et al., Cancer Res 51: 3281-3288, 1991). Here we report responses that occur in these cells downstream of PKC activation or downregulation. We examined induction of 12-O-tetradecanoylphorbol-13-acetate (TPA)-inducible sequence (TIS) gene expression in Mel-ab melanocytes and in their transformed counterparts, B16 melanoma cells. Exposure of quiescent Mel-ab cells to the PKC-activating phorbol esters TPA or sapintoxin A at 81 nM for 2 h increased levels of mRNA for six of seven TIS genes examined (twofold to 80-fold increase in steady-state RNA levels for TIS 1, 7, 8, 11, 21, and 28 (c-fos); TIS 10 expression was not affected). No induction of TIS gene expression was observed either in growing Mel-ab cells maintained in 324 nM phorbol 12,13-dibutyrate or in B16 cells previously unexposed to phorbol esters, in which normal PKC levels were endogenously depressed. The cAMP-elevating agents choleratoxin (10 nM) and dibutyryl cyclic AMP (2.5 mM) increased levels of TIS mRNA (with the exception of TIS 10) in both proliferating Mel-ab and B16 cells, suggesting that downregulation of the PKC pathway is specific and not a consequence of a general inhibition of all signalling pathways.

3T3 Cells↗
PubMed1992

Cell and tissue responses of a murine tumour to phthalocyanine-mediated photodynamic therapy.

Mice bearing a subcutaneously growing tumour (Colo 26) were injected intravenously with the photosensitiser chloroaluminum sulphonated phthalocyanine (5 mg/kg) 24 h prior to irradiating the tumour with laser light (675 nm; 50mW, 100 J/tumour). Energy status of the tumour, as assessed by the loss of high energy phosphates in the 31P-nuclear magnetic resonance spectra, was altered dramatically following treatment, such that the ATP fell to undetectable levels within 1 h of light irradiation. However, assessment of the clonogenic capacity of neoplastic cells isolated from dissociated tumours showed that these rapid changes in cellular metabolism were not reflected in similar rapid changes in cell viability. Reductions in clonogenic capacity, which fell to less than 0.1% of control values at 24h postirradiation, closely mirrored those resulting from the cessation of vascular perfusion. Evaluation of tumour blood flow, using the technique of hydrogen washout, showed that the treatment protocol evoked a gradual and selective reduction in flow within the tumour resulting in complete vascular stasis by approximately 5 h after treatment. The results indicate that while chloroaluminum sulphonated phthalocyanine-mediated photodynamic therapy caused early metabolic damage in neoplastic cells, loss of viability paralleled the induction of complete inhibition of vascular flow in the tumour.

Animals↗
PubMed1992

Inhibitors of nitric oxide synthase selectively reduce flow in tumor-associated neovasculature.

1. The effects of L-arginine analogues, NG-nitro-L-arginine methyl ester (L-NAME) and NG-monomethyl-L-arginine (L-NMMA) and methylene blue on blood flow in a murine adenocarcinoma and melanoma have been investigated. 2. Sponge implants in Balb/c and C57/BL mice were used to host proliferating tumour cells while the washout of 133Xe was employed to assess local blood flow in the implanted sponges. 3. Pharmacological inhibition of nitric oxide (NO) reduced blood flow in both tumours but this effect was reversed by administration of L-arginine. 4. In marked contrast, the effect of these same NO inhibitors on the blood flow in sponge-induced non-neoplastic granulation tissue was negligible. 5. These results strongly suggest that: (a) flow in tumour vessels is modulated by nitric oxide which maintains a dilator tone in neoplastic tissue; (b) the constrictor activity (as monitored by an increase in t1/2 of 133Xe) of NO inhibitors may be attributed to the removal of such dilator tone; (c) many of the abnormalities described in tumour vasculature, such as hyporeactivity or unresponsiveness to vasoactive mediators and maximum vasodilation, may be due to an increase in NO synthesis in cancers.

Adenocarcinoma↗
PubMed1991

Isolation and characterization of human melanoma cell variants expressing high and low levels of CD44.

Variants of the human melanoma cell line LT5.1 were selected for high and low expression of the Mr 90,000 CD44 glycoprotein by using the Hermes-3 monoclonal antibody combined with fluorescence-activated cell sorting. Cells were single cell cloned and clones of CD44 high-expressing and CD44 low-expressing phenotype were isolated. The variants, which exhibited up to a 7-fold difference between high and low expression, have maintained a stable phenotype over a period of 3 months in tissue culture. Northern blot analysis of mRNA from the different clones showed correlation of levels of transcripts with fluorescence-activated cell sorting analysis data. Wound migration assays, utilizing the different clones, showed that the low-expressing clones manifested less motility than did cells showing high levels of CD44. Homotypic aggregation of cells was increased in those cells expressing high levels of CD44, and these variants were also better able to adhere to hyaluronate substrates. All of these activities were inhibited by the presence of anti-CD44 antibody. When injected i.v. into nu/nu BALB/c mice, the low-expressing clones gave significantly fewer lung nodules than the high-expressing clones, although the two variant types did not differ in their capacity to form s.c. tumors in similar mice. These results suggest that the CD44 molecule, possibly as a function of its activities as a hyaluronate receptor, may play a vital role in determining the fate of hematogenously disseminating melanoma cells.

Animals↗
PubMed1991

Integrin expression in human melanoma cell lines: heterogeneity of vitronectin receptor composition and function.

Ten human melanoma cell lines were examined for integrin-receptor expression using a panel of antibodies directed against different integrin subunits. Considerable heterogeneity was detected for levels of expression of 7 integrins, including the alpha v beta 3 vitronectin receptor where a correlation between tumorigenic capacity in athymic nude mice and alpha v beta 3 levels was found. Detailed analysis of the vitronectin receptor on these lines revealed heterogeneity of composition. In one cell line, VUP, an alpha v beta 1 association was detected and, by antibody-inhibition studies, this receptor was shown to bind vitronectin as its ligand. In another line, DX3, evidence was obtained which indicated that apart from the alpha v beta 3 receptor the alpha v was able to associate with another beta subunit which was not beta 3. The existence of these alternative forms of the vitronectin receptor in this small sample of tumours of common origin might explain why the capacity to bind to fibrinogen and vitronectin substrates by these cells did not necessarily correlate with alpha v beta 3 levels.

Animals↗