Biomedical subjects

C J Wraight

Publications and source records attributed to C J Wraight.

18 recordsLinked to original sources

PubMed2006

A GH receptor antisense oligonucleotide inhibits hepatic GH receptor expression, IGF-I production and body weight gain in normal mice.

Diabetic retinopathy and acromegaly are diseases associated with excess action of GH and its effector IGF-I, and there is a need for improved therapies. We have designed an optimised 2'-O-(2-methoxyethyl)-modified phosphorothioate oligodeoxynucleotide, ATL 227446, and demonstrated its ability to suppress GH receptor mRNA in vitro. Subcutaneous injections of ATL 227446 reduced GH receptor mRNA levels, GH binding activity and serum IGF-I levels in mice after seven days of dosing. The reduction in serum IGF-I could be sustained for over ten weeks of dosing at therapeutically relevant levels, during which there was also a significant decrease in body weight gain in antisense-treated mice relative to saline and mismatch control-treated mice. The findings indicate that administration of an antisense oligonucleotide to the GH receptor may be applicable to human diseases in which suppression of GH action provides therapeutic benefit.

Animals↗

PubMed2005

Insulin-like growth factor binding protein-3 (IGFBP-3) localizes to and modulates proliferative epidermal keratinocytes in vivo.

BACKGROUND: The colocalization of insulin-like growth factor binding protein-3 (IGFBP-3) and IGF-I receptor (IGF-IR) in the basal/germinative layer of the epidermis suggests a key role in modulating epidermal homeostasis. OBJECTIVES: We aimed to clarify both the specific cellular localization and the effect of excess epidermal IGFBP-3 on keratinocyte proliferation. METHODS: (i) Total RNA was isolated from fluorescence-activated cell sorted basal human keratinocyte subtypes [keratinocyte stem cells, transit amplifying keratinocytes (TA), postmitotic differentiating keratinocytes (PMD)], and real-time polymerase chain reaction analysis was used to determine the abundance of IGFBP-3 and IGF-IR mRNAs. (ii) An IGFBP-3 transgenic mouse model was then used to assess the effect of excess epidermal IGFBP-3 on keratinocyte proliferation. Excess epidermal IGFBP-3 mRNA and protein was determined by in situ hybridization and immunohistochemistry, respectively. RESULTS: (i) The highest levels of IGFBP-3 mRNA were detected in TA keratinocytes, in contrast to IGF-IR mRNA levels which were highest in PMD keratinocytes. (ii) Elevated human IGFBP-3 mRNA and protein was confirmed in the epidermis of skin derived from transgenic mice. Excess IGFBP-3 reduced the relative percentage of proliferative keratinocytes (Ki67 positive) irrespective of skin location (belly, back and tail). Thus, in the epidermis, IGFBP-3 mRNA is highly expressed by proliferative keratinocytes (TA) and overexpression of IGFBP-3 inhibits keratinocyte proliferation. CONCLUSIONS: We conclude that in vivo IGFBP-3 ensures epidermal homeostasis via downregulation of keratinocyte proliferation, and thus modulates the early stages of keratinocyte differentiation.

Adult↗

PubMed2004

Antisense oligonucleotide treatments for psoriasis.

Antisense oligonucleotides are emerging as an exciting therapeutic strategy for treating skin diseases such as psoriasis. Potential antisense targets are proteins upregulated in psoriatic skin, in particular those associated with inflammation (intercellular adhesion molecule [ICAM]-1, IL-2 and -8), proliferation (insulin-like growth factor type I receptor [IGF-IR], epidermal growth factor) and hyperangiogenesis (vascular endothelial growth factor [VEGF]). Whereas topical application and subsequent penetration of large oligonucleotides into normal skin is problematic, the impaired barrier function of psoriatic lesions permits the uptake of antisense drugs. Studies to date indicate that topically applied antisense molecules can be delivered to target cells in the epidermis and dermis of psoriatic skin. Antisense-mediated suppression of target mRNA and protein has been demonstrated in models of human skin grafted to immunosuppressed mice and in hairless mouse models of skin inflammation. In a xenograft model of human psoriasis, treatment with repeated intradermal injections of IGF-IR antisense caused a normalisation of the epidermal hyperproliferation. This class of drug, therefore, holds much potential for the successful treatment of psoriasis in the clinical setting.

Animals↗

PubMed2001

Antisense oligonucleotides in cutaneous therapy.

Antisense oligonucleotides have been the subject of intense interest as research tools to elucidate the functions of gene products and as therapeutic agents. Initially, their mode of action was poorly understood and the biological effects of oligonucleotides were often misinterpreted. However, research into these gene-based inhibitors of cellular action recently has succeeded in realising their exciting potential, particularly as novel therapeutic agents. An emerging application of this technology is in cutaneous therapy. The demand for more effective dermatological drugs will ensure further development of antisense strategies in skin, with key issues being drug delivery, therapeutic target selection, and clinical applicability.

Antineoplastic Agents↗

PubMed2001

Calcium regulates the expression of insulin-like growth factor binding protein-3 by the human keratinocyte cell line HaCaT.

The insulin-like growth factor (IGF) system is essential for epidermal homeostasis. Insulin-like growth factor binding protein 3 (IGFBP-3), a modulator of IGF action that also exhibits IGF-independent activity, is localized to selected keratinocytes in the basal epidermal layer and may thus contribute to keratinocyte differentiation. We have utilized the human keratinocyte cell line, HaCaT, to examine the effect of calcium on the regulation of components of the IGF system. Western ligand and northern blot analyses revealed secreted IGFBP-3 and IGFBP-3 mRNA were reduced by an elevation in calcium levels in the culture medium. At 1.0 and 1.2 mM CaCl2 culture conditions IGFBP-3 abundance was reduced to 36% +/- 1.6% and 26% +/- 7.1%, respectively, of that from cells grown at 0.03 mM CaCl2. IGFBP-3 mRNA levels in 0.7 mM and 1.2 mM CaCl2 were reduced to 46% +/- 17.4% and 24% +/- 4.6%, respectively, compared with IGFBP-3 mRNA levels at 0.03 mM CaCl2. The observed reduction of IGFBP-3 was not associated with IGFBP-3 proteolysis. In contrast IGF-I receptor protein and mRNA levels remained unchanged. The IGF-I stimulated proliferative response of HaCaT keratinocytes showed that under low (0.03 mM) and high (1.2 mM) CaCl2 conditions IGF-I at 100 and 1000 ng per ml similarly increased cell number 2.4- and 2.7-fold, respectively, with similar dose-response curves. HaCaT keratinocytes grown under medium (0.7 mM) and high (1.2 mM), but not low (0.03 mM), CaCl2 conditions for 21 d revealed an induction of profilaggrin mRNA, a marker of keratinocyte differentiation. These studies indicate that the exposure of HaCaT keratinocytes to elevated calcium levels is associated with a decline in IGFBP-3 but not IGF-I receptor levels. These findings suggest a potential mechanism for the distribution of IGFBP-3 in the epidermis, which may be involved in the process of keratinocyte differentiation.

Calcium↗

PubMed2000

Reversal of epidermal hyperproliferation in psoriasis by insulin-like growth factor I receptor antisense oligonucleotides.

Epidermal hyperplasia is a key feature of the common skin disorder psoriasis. Stimulation of epidermal keratinocytes by insulin-like growth factor I (IGF-I) is essential for cell division, and increased sensitivity to IGF-I may occur in psoriasis. We hypothesized that inhibition of IGF-I receptor expression in the psoriasis lesion would reverse psoriatic epidermal hyperplasia by slowing the rate of keratinocyte cell division. Here we report the use of C5-propynyl-dU,dC-phosphorothioate antisense oligonucleotides to inhibit IGF-I receptor expression in keratinocytes. We identified several inhibitory antisense oligonucleotides and demonstrated IGF-I receptor inhibition in vitro through an mRNA targeting mechanism. Repeated injection of these oligonucleotides into human psoriasis lesions, grafted onto nude mice, caused a dramatic normalization of the hyperplastic epidermis. The findings indicate that IGF-I receptor stimulation is a rate-limiting step in psoriatic epidermal hyperplasia and that IGF-I receptor targeting by cutaneous administration of antisense oligonucleotides forms the basis of a potential new psoriasis therapy.

Animals↗

PubMed2000

Antisense inhibition of IGF receptor expression in HaCaT keratinocytes: a model for antisense strategies in keratinocytes.

Antisense strategies targeting skin conditions are attractive in concept, with a number of possible pathologic conditions, such a psoriasis, apparently suitable for such an approach. Because in vitro screening of candidate sequences is usually desirable, we have attempted to use a range of new generation cationic lipids to produce significant antisense oligodeoxynucleotide (ODN) uptake in an immortalized keratinocyte cell line (HaCaT). A large number of commercially available lipids were screened for the ability to induce nuclear ODN localization: Tfx-50, Tfx-20, Tfx-10, Superfect, Cytofectin GSV, Perfect lipids 1-8, Lipofectin, and Lipofectamine. All lipids were used at a range of concentrations (1-20 microg/ml) and with a range of ODN concentrations (1-1000) nM). Of all lipids used, only Cytofectin GSV and Superfect produced significant (>30% of cells) levels of nuclear positive cells, with Superfect also producing significant toxicity at the effective concentration used. Only two treatments produced a significant reduction in target mRNA: insulin-like growth factor-1 receptor (IGF-1R)-ODN 64 complexed with Cytofectin GSV (27.1% +/- 3.5% of IGF-1R mRNA in untreated cells,p < 0.01) and ODN 64 complexed with 10 microg/ml Lipofectin (62.2% +/- 3.4% of IGF-1R mRNA in untreated cells, p < 0.05). Only one treatment, ODN 64 complexed with Cytofectin GSV, produced a reduction in cell growth and survival as assessed by amido black assay. These results demonstrate that in HaCaT keratinocytes, Cytofectin GSV alone of all commercially available cationic lipids was effective in delivering antisense ODN into cell nuclei such that a profound antisense effect could be demonstrated.

Cell Line, Transformed↗

PubMed1999

Expression of insulin-like growth factor binding protein-3 (IGFBP-3) in human keratinocytes is regulated by EGF and TGFbeta1.

Insulin-like growth factor-I (IGF-I) is essential for normal epidermal homeostasis; however, the role of IGF binding proteins (IGFBPs), regulators of IGF action, remains unclear. Here we examine the regulation of human keratinocyte-produced IGFBPs by epidermal growth factor (EGF), transforming growth factor beta 1 (TGFbeta1), and IGF-I, growth factors known to be active in skin. In the absence of added growth factors, IGFBP-3 was the major binding protein secreted into the medium by primary keratinocytes. Addition of EGF or TGFbeta1 to keratinocyte cultures resulted in a significant decrease in IGFBP-3 abundance in conditioned medium when compared with control, untreated cells. Specifically, EGF (50 ng/ml) and TGFbeta1 (50 ng/ml) reduced IGFBP-3 abundance to 15+/-6% and 22+/-9%, respectively. Using Northern blot analysis, we found EGF and TGFbeta1 (50 ng/ml) to reduce IGFBP-3 mRNA levels in keratinocytes to 51+/-12% and 50+/-38%, respectively, when compared with control, untreated cells. Treatment with IGF-I or its analogue des(1-3)IGF-I did not lead to any consistent change in IGFBP-3 abundance. However, both IGF-I and des(1-3)IGF-I at 100 ng/ml led to a modest increase in IGFBP-3 mRNA levels in keratinocytes, suggesting posttranscriptional regulation of IGFBP-3 abundance. We propose that local modulation of IGFBP-3 abundance may represent another level of regulation of growth factor action in the epidermis, where EGF and TGFbeta1 and possibly other local growth factors specifically regulate the availability of IGF-I to its keratinocyte receptors.

Culture Media, Conditioned↗

PubMed1999

Oligonucleotide uptake in cultured keratinocytes: influence of confluence, cationic liposomes, and keratinocyte cell type.

The success of anti-sense strategies has been limited, at least in part, by the poor uptake of these agents into the target cells. In keratinocytes, there is conflicting evidence as to the amount and location of oligonucleotide uptake into these cells, with variable proportions of cells reported to take up oligodeoxynucleotide, and also cytoplasmic and nuclear localization reported. In this study, the uptake of oligodeoxynucleotides in cultured normal human keratinocytes and the HaCaT cell line was quantitated in the presence of various lipids designed to enhance uptake and in varying culture conditions. About 12% of cells in a confluent normal human keratinocyte culture showed nuclear uptake, with a small and variable proportion showing cytoplasmic localization after 24 h incubation with 1 microM oligodeoxynucleotide. Uptake of oligodeoxynucleotide was found to be increased by liposome encapsulation (to a maximum of 28.1% +/- 2.1% of cells), low confluence (39.5% +/- 2.5%), and further increased by a combination of the two conditions (55.4% +/- 4.3%). HaCaT cell populations showed sparse but consistent uptake of oligodeoxynucleotide, with about 1% of cells showing nuclear localization in the presence of 1 microM oligodeoxynucleotide, increasing to 13.5% +/- 4.9% in the presence of cationic lipid (Tfx-50) in low confluence HaCaT monolayers. We conclude that normal keratinocytes exhibit reliable, substantial uptake of oligonucleotides in conditions controlled for confluence and aided by liposome encapsulation.

Adult↗

PubMed1999

Live confocal microscopy of oligonucleotide uptake by keratinocytes in human skin grafts on nude mice.

Anti-sense oligonucleotide uptake by keratinocytes in human skin grafts on athymic mice was examined using live confocal microscopy. Fluorescein isothiocyanate-labeled 15-mer C-5 propyne modified phosphorothioate anti-sense oligonucleotide (10-50 microM) was intradermally injected into normal human skin grafts on athymic mice, and the localization of the anti-sense oligonucleotide was assessed after 1-24 h postinjection. Anti-sense oligonucleotide was found to localize in the nuclei of basal and suprabasal keratinocytes after 1-2 h, and this localization was still observed after 24 h. This live in vivo observation of anti-sense oligonucleotide uptake in basal keratinocytes was confirmed using conventional fluorescence microscopy of fixed sections of skin grafts. Neither single nucleotides which were fluorescein isothiocyanate-labeled nor fluorescein isothiocyanate alone was able to penetrate into the nuclei of human skin graft keratinocytes after intradermal injection, and hence it is likely that the anti-sense oligonucleotide was not degraded prior to intracellular localization. Topical administration of anti-sense oligonucleotide and anti-sense oligonucleotide-liposome complexes resulted primarily in localization in the stratum corneum of human skin grafts. When grafts were tape stripped prior to anti-sense oligonucleotide administration, however, as little as 5 microM anti-sense oligonucleotide was required to observe nuclear anti-sense oligonucleotide accumulation. These results suggest that cutaneous anti-sense strategies can be tested using delivery via intradermal anti-sense oligonucleotide injection in human skin grafts on athymic mice, and that agents providing penetration of anti-sense oligonucleotide across the stratum corneum are likely to be required for successful topical therapies.

Administration, Topical↗

PubMed1999

Interactions between growth hormone, insulin-like growth factor I, and basic fibroblast growth factor in melanocyte growth.

Melanocytes, highly differentiated neural crest-derived cells, are located in the basal layer of the epidermis, where they play a role in protecting against UV damage in the skin. Previous studies suggest that both growth hormone (GH) and the insulin-like growth factor I (GH/IGF-I) system may be important for melanocyte growth and function. We have therefore characterized the role of the GH/IGF system in melanocyte growth in vitro and its interaction with the local growth factor basic fibroblast growth factor (bFGF). Analysis of the effects of GH, IGF-I, and bFGF and combinations of these growth factors on melanocyte growth in vitro revealed that 1) GH stimulates the growth of melanocytes when combined with IGF-I, des(1-3)IGF-I [an analog of IGF-I that has a reduced binding affinity for IGF-binding proteins (IGFBPs)], or bFGF, either separately or in combination; 2) in contrast to the lack of effect of GH or bFGF alone, both IGF-I and des(1-3)IGF-I enhance melanocyte growth in a dose-dependent manner; and 3) IGF-I is more efficacious in eliciting a growth response at low concentrations compared to des(1-3)IGF-I. Using Western ligand blotting, affinity cross-linking, immunoprecipitation, RIA, and Northern analysis, we show that cultured human melanocytes synthesize and secrete minimal amounts of IGFBP. IGFBP-4 is the major IGFBP produced by these cells when cultured in complete growth medium or in the presence of either IGF-I or des(1-3)IGF-I alone. In conclusion, these studies provide support for a role for both GH and IGF-I in the growth of human melanocytes in vitro, involving synergy with bFGF. Low levels of melanocyte-derived IGFBP-4 may play a role in enhancing the modulation of IGF action.

Blotting, Northern↗

PubMed1998

Intranuclear localization of insulin-like growth factor binding protein-3 (IGFBP-3) during cell division in human keratinocytes.

Insulin-like growth factor-I (IGF-I) stimulation of basal keratinocytes is an essential component of normal epidermal homeostasis. In addition to the IGF receptor, basal keratinocytes synthesize insulin-like growth factor binding protein-3 (IGFBP-3). The HaCaT keratinocyte cell line, which has many characteristics of basal keratinocytes, synthesizes IGFBP-3 that in vitro reduces its IGF-I responsiveness. IGFBP-3 has attracted interest as a potential growth arrest protein, both via its ability to modulate IGF-I responsiveness, and more controversially via IGF-I-independent mechanisms. Intracellular modes of action have been proposed, and a nuclear localization consensus sequence has previously been identified within IGFBP-3. Using immunocytochemistry with a biotinylated antibody specific for IGFBP-3, we investigated the intracellular localization of IGFBP-3 in subconfluent monolayer cultures of HaCaT cells. Diffuse cellular staining was visible, potentially corresponding to cell surface and nascent cytoplasmic IGFBP-3. Of particular interest however, was the localization of staining over the nuclei of a large proportion of cells that were undergoing cell division. Antibody staining was specific for IGFBP-3 because addition of recombinant human IGFBP-3 to the antibody prior to incubation with the cells inhibited these staining patterns. Optical sections obtained using a confocal laser scanning microscope showed that in keratinocytes undergoing cell division, IGFBP-3 was localized inside the nucleus. These results show that intracellular IGFBP-3 localization is altered during the cell cycle and suggest a possible nuclear role for IGFBP-3 during cell division.

Cell Division↗

PubMed1997

Expression of insulin-like growth factor binding protein-3 (IGFBP-3) in the psoriatic lesion.

Epidermal hyper-proliferation is a key feature of psoriasis, and a role for IGF-I in this process has previously been proposed. Herein we investigated the expression of IGF binding proteins in the psoriatic lesion and compared it with normal skin. With in situ hybridization, we found that IGFBP-3 mRNA was expressed in the basal layer of the epidermis in normal skin. IGFBP-3 was also detected immunohistochemically, exclusively in the basal layer. In the psoriatic lesion, IGFBP-3 mRNA was similarly limited to the basal layer despite the characteristic expansion of the basaloid keratinocyte compartment and was detected only in the suprapapillary epidermis, where IGFBP-3 mRNA was more abundant than in normal or uninvolved epidermis, and IGFBP-3 protein could be readily detected with specific antibody. As with IGFBP-3 mRNA, which represents the likely site of IGFBP-3 synthesis, IGFBP-3 was strictly limited to the basal keratinocytes of the suprapapillary epidermis. By using an antibody to the cell cycle antigen Ki67, we also showed that the suprapapillary epidermis, where IGFBP-3 expression was maximal, contained few keratinocytes undergoing mitosis, whereas the tips of the rete pegs, where IGFBP-3 expression was conspicuously absent, contained many keratinocytes undergoing mitosis. We suggest that IGFBP-3 is a growth inhibitor in basal keratinocytes and that absence of IGFBP-3 in the tips of rete pegs may contribute to epidermal hyper-proliferation in the psoriatic lesion.

Adult↗

PubMed1995

Identification of insulin-like growth factor binding proteins from cultured human epidermal keratinocytes.

The role and mechanisms of action of insulin-like growth factors (IGFs) in skin remain unclear. Epidermal keratinocytes possess IGF-I receptors and are responsive to IGF-I, which is primarily derived from underlying dermal fibroblasts. IGF binding proteins (IGFBPs), also synthesized by fibroblasts, may be involved in paracrine targeting of IGF-I to its receptors. We therefore examined whether human keratinocytes synthesize IGFBPs and their mRNAs. Following culture in complete medium (containing bovine pituitary extract and epidermal growth factor) Western ligand blotting (WLB) of cell conditioned medium revealed a major band of 32 kD, a less abundant IGFBP of 24 kD at all passages, and a 37-42 kD IGFBP which increased in abundance in late passage. Immunoprecipitation followed by WLB confirmed that the predominant 32 kD band was IGFBP-2. Radioimmunoassay of IGFBP-1, -3, and -6 revealed detectable levels of IGFBP-3 and significant levels of IGFBP-6, but not IGFBP-1. Northern analysis following culture in complete medium revealed that at early passage IGFBP-1, -2, -4, and -6 mRNAs were detectable. IGFBP-3 and -5 mRNAs were not detectable. Following culture in growth factor-free medium a 37-42 kD band, consistent with IGFBP-3, was predominant and a 24 kD band consistent with IGFBP-4 was also present. These data demonstrate the expression of a distinct pattern of IGFBPs by cultured human keratinocytes dependent on culture conditions. Keratinocyte-derived IGFBPs are likely to play a role in the transport and targeting of IGF-I from dermally derived fibroblasts to the epidermis.

Blotting, Northern↗

PubMed1995

Insulin-like growth factor-I and epidermal growth factor regulate insulin-like growth factor binding protein-3 (IGFBP-3) in the human keratinocyte cell line HaCaT.

The human keratinocyte cell line HaCaT has a basal phenotype and secretes an insulin-like growth factor (IGF) binding protein, IGFBP-3, which modulates its IGF-I response. Keratinocytes are highly responsive to mitogenic stimulation by IGF-I and epidermal growth factor (EGF), but the effect of these growth factors on IGFBP secretion by keratinocytes is not known. We investigated the effects of IGF-I and EGF, as well as three other skin-growth regulators, retinoic acid, basic fibroblast growth factor, and dexamethasone, on mitogenic stimulation and IGFBP-3 production in HaCaT cells. IGF-I and EGF were strongly mitogenic, whereas retinoic acid, basic fibroblast growth factor, and dexamethasone were not significantly mitogenic. IGF-I increased the level of IGFBP-3 in cell-conditioned medium by up to twofold, whereas EGF caused a twentyfold reduction in IGFBP-3. Retinoic acid and basic fibroblast growth factor had only minor effects on IGFBP-3 and dexamethasone had no effect. IGF-I stimulation of IGFBP-3 did not involve increases in IGFBP-3 mRNA; however, EGF, consistent with its effect on IGFBP-3 protein, caused a fivefold reduction in IGFBP-3 mRNA. In summary, EGF profoundly inhibited IGFBP-3 synthesis in basal keratinocytes, whereas IGF-I increased IGFBP-3 levels by a posttranscriptional mechanism. We hypothesize that by inhibiting IGFBP-3 production in basal keratinocytes, epidermal mitogens such as EGF might stimulate epidermal growth indirectly by increasing local IGF-I availability.

Cell Division↗

PubMed1994

A keratinocyte cell line synthesizes a predominant insulin-like growth factor-binding protein (IGFBP-3) that modulates insulin-like growth factor-I action.

Insulin-like growth factor-I (IGF-I) is an important regulator of epidermal proliferation and has been shown in vitro to be a powerful stimulator of keratinocyte growth. It is synthesized by fibroblasts in the dermis, along with several IGF-binding proteins (IGFBPs), which are known to modulate IGF-I responsiveness of virtually all tissues studied. Because it was not known how or in what form IGF-I produced in the dermis acts on epidermal keratinocytes in vivo, we investigated the possible role of IGFBPs in modulating the response of epidermal keratinocytes to IGF-1. We show here that tIGF-I, a non-IGFBP-binding analogue of IGF-1, is a more potent mitogenic stimulator of the keratinocyte cell line HaCaT than IGF-I, suggesting that keratinocytes produce IGFBPs that modulate their response to IGF-I. To confirm this and to identify which IGFBPs were produced, we analyzed HaCaT cell-conditioned medium and mRNA, with the following findings: HaCaT cells produce a major IGFBP, identified as IGFBP-3, and a minor 24-kD IGFBP, likely to be IGFBP-4. Northern analysis revealed a 2.6-kb IGFBP-3 mRNA; however, IGFBP-4 mRNA was not detectable. We conclude that production of predominantly IGFBP-3 by the HaCaT cell line modulates its sensitivity to IGF-I stimulation. Epidermal IGFBPs thus have a potential role in vivo in the interaction of dermis derived IGF-I with epidermal keratinocytes.

Blotting, Western↗

PubMed1990

Human major histocompatibility complex class II invariant chain is expressed on the cell surface.

Class II major histocompatibility complex antigens are intracellularly associated with a nonpolymorphic polypeptide referred to as the invariant chain. Before the class II heterodimer appears on the cell surface, the invariant chain dissociates but it has so far been unclear as to whether or not a proportion of the invariant chain also appears on the plasma membrane. We describe a study with three monoclonal antibodies which recognize an extracytoplasmic determinant present on all forms of the invariant chain and use them to demonstrate its presence on the surface of the intact cells. The determinants recognized by two of the antibodies were found to be located within the 60 amino acids at the extreme C-terminal (extracytoplasmic) end of the invariant chain. The invariant chain-specific monoclonal antibody, VIC-Y1, was found to bind a determinant located between amino acids 1 and 73, which correspond to mainly cytoplasmic residues. Using the C-terminal specific antibodies, the number of antibody binding sites on the surface of two B lymphoma lines was estimated to be 10(5) per cell. The results of this study appear to resolve the highly disputed question of whether or not the invariant chain can appear as a plasma membrane protein. The results are discussed in the context of a possible role for the invariant chain in antigen processing and presentation.

Antibodies, Monoclonal↗

PubMed1986

The effect of pristane on ascites tumor formation and monoclonal antibody production.

Preinjection of mice 10 days before intraperitoneal injections of hybridomas gives optimum development of ascites tumors with respect to the rate at which tumors form, percentage of mice developing tumors, volume of ascites fluid produced, and concentration of monoclonal antibody in ascites fluid. The dose of pristane injected has little or no effect on ascites formation down to 0.1 ml of pristane.

Animals↗