Quantitative immunohistochemistry by measuring cumulative signal strength using commercially available software photoshop and matlab.

Currently available techniques for performing quantitative immunohistochemistry (Q-IHC) rely upon pixel-counting algorithms and therefore cannot provide information as to the absolute amount of chromogen present. We describe a novel algorithm for true Q-IHC based on calculating the cumulative signal strength, or energy, of the digital file representing any portion of an image. This algorithm involves subtracting the energy of the digital file encoding the control image (i.e., not exposed to antibody) from that of the experimental image (i.e., antibody-treated). In this manner, the absolute amount of antibody-specific chromogen per pixel can be determined for any cellular region or structure. (J Histochem Cytochem 48:303-311, 2000)     

Expression of GRP and its receptor in well-differentiated colon cancer cells correlates with the presence of focal adhesion kinase phosphorylated at tyrosines 397 and 407.

Gastrin-releasing peptide (GRP) and its receptor (GRP-R) are not normally expressed by epithelial cells lining the colon but are aberrantly expressed in cancer, where they act as morphogens and regulate tumor cell differentiation. Studies of colon cancer formation in mice genetically incapable of synthesizing GRP-R suggested that this receptor's morphogenic properties were mediated via focal adhesion kinase (FAK). We therefore set out to determine the presence of both total and phosphorylated forms of FAK in human colon cancer specimens as a function of tumor cell differentiation and GRP/GRP-R co-expression. Ten colon cancers containing 25 regions of distinct differentiation were randomly selected from our GI Cancer Tumor Bank. All specimens were immunohistochemically probed using antibodies recognizing GRP, GRP-R, total FAK, and FAK specifically phosphorylated at tyrosine (Y) 397, 407, 576, 577, 861, and 925. Antibody-specific chromogen was determined by quantitative immunohistochemistry (IHC) for each region of defined differentiation. Here we confirm that GRP/GRP-R co-expression is a function of differentiation, with highest levels observed in well-differentiated tumor cells. We also show that the amount of total FAK and of FAK phosphorylated at Y397 and Y407 tightly correlates with differentiation and with the amount of GRP/GRP-R co-expression. These findings are consistent with GRP/GRP-R acting as a morphogen by activating FAK, and suggest that this occurs via phosphorylation of this enzyme at two specific tyrosine residues.     

Consequence of gastrin-releasing peptide receptor activation in a human colon cancer cell line: a proteomic approach

Gastrin-releasing peptide (GRP) and its receptor (GRPR) are aberrantly up-regulated in colon cancer. When expressed, they act as morphogens, retaining tumor cells in a better differentiated state and retarding metastasis. To identify targets activated in response to GRPR signaling we studied Caco-2 and HT-29 cells, colon cancer cell lines that expresses GRPR as a function of confluence. Total cell protein was extracted from pre-confluent cells (expressing GRP/GRPR) cultured in serum-free media in the presence or absence of GRPR-specific antagonist; as well as from confluent cells that do not express GRPR. Overall, we identified 5 proteins that are specifically down-regulated after GRP/GRPR expression: Bach2, creatine kinase B, p47, and two that could not be identified; and 6 proteins that are up-regulated: gephyrin, HSP70, HP1, ICAM-1, ACAT, and one that could not be identified. These findings suggest that the mechanism(s) by which GRP/GRPR mediate its morphogenic effects in colon cancer involve the actions of a number of hitherto unappreciated proteins.     

Effects of bombesin on human small cell lung cancer cells: evidence for a subset of bombesin non-responsive cell lines

The effects of bombesin on three human small cell lung carcinoma cell (SCLC) lines (NCI-H69, NCI-H128, and NCI-H345) have been examined and compared to the effects of the peptide on the mouse fibroblast cell line Swiss 3T3, and the rat pituitary tumor cell line GH3W5. While all three SCLC lines expressed messenger RNA encoding pro-gastrin releasing peptide (GRP), only the NCI-H345 cells expressed detectable membrane receptors for GRP and responded to nanomolar concentrations of bombesin as shown by 125I-GRP binding, total inositol phosphate accumulation, and increased clonal growth in soft agarose. These data show that some SCLC lines are insensitive to bombesin and do not express detectable membrane receptors for GRP.     

Gastrin-releasing peptide and cancer

Over the past 20 years, abundant evidence has been collected to suggest that gastrin-releasing peptide (GRP) and its receptors play an important role in the development of a variety of cancers. In fact, the detection of GRP and the GRP receptor in small cell lung carcinoma (SCLC), and the demonstration that anti-GRP antibodies inhibited proliferation in SCLC cell lines, established GRP as the prototypical autocrine growth factor. All forms of GRP are generated by processing of a 125-amino acid prohormone; recent studies indicate that C-terminal amidation of GRP18-27 is not essential for bioactivity, and that peptides derived from residues 31 to 125 of the prohormone are present in normal tissue and in tumors. GRP receptors can be divided into four classes, all of which belong to the 7 transmembrane domain family and bind GRP and/or GRP analogues with affinities in the nM range. Over-expression of GRP and its receptors has been demonstrated at both the mRNA and protein level in many types of tumors including lung, prostate, breast, stomach, pancreas and colon. GRP has also been shown to act as a potent mitogen for cancer cells of diverse origin both in vitro and in animal models of carcinogenesis. Other actions of GRP relevant to carcinogenesis include effects on morphogenesis, angiogenesis, cell migration and cell adhesion. Future prospects for the use of radiolabelled and cytotoxic GRP analogues and antagonists for cancer diagnosis and therapy appear promising.     

Selective targeting and inducible destruction of human cancer cells by retroviruses with envelope proteins bearing short peptide ligands

In the accompanying study, we show how retroviral tropism can be redirected by insertion of short peptide ligands at multiple locations in envelope. Here we use this approach to selectively target and destroy human cancer cells. Many cancer cells overexpress specific cell surface receptors. We have generated Moloney murine leukemia virus (MLV) envelope derivatives bearing short peptide ligands for gastrin-releasing protein (GRP) and human epidermal growth factor receptors. Pseudotyped viruses containing these chimeric envelope derivatives selectively transduce human cancer cell lines that overexpress the cognate receptor. A retrovirus targeting the GRP receptor can deliver the thymidine kinase gene to human melanoma and breast cancer cells, which are killed by the subsequent addition of ganciclovir. Collectively, our results demonstrate that short peptide ligands inserted at appropriate locations in MLV envelope can selectively target retroviruses to human cancer cells and deliver a therapeutically relevant gene.     

Transient upregulation of GRP and its receptor critically regulate colon cancer cell motility during remodeling

Gastrin-releasing peptide (GRP) is typically viewed as a growth factor in cancer. However, we have suggested that in colon cancer, GRP acts primarily as a morphogen when it and its receptor (GRP-R) are aberrantly upregulated. As such, GRP/GRP-R act(s) primarily to modulate processes contributing to the assumption or maintenance of tumor differentiation. One of the most important such processes is the ability of tumor cells to achieve directed motility in the context of tissue remodeling. Yet the cellular conditions affecting GRP/GRP-R expression, and the biochemical pathways involved in mediating its morphogenic properties, remain to be established. To study this, we evaluated the human colon cancer cell lines Caco-2 and HT-29 cells. We found that confluent cells do not express GRP/GRP-R. In contrast, disaggreation and plating at subconfluent densities results in rapid GRP/GRP-R upregulation followed by their progressive decrease as confluence is achieved. GRP/GRP-R coexpression correlated with that of focal adhesion kinase (FAK) phosphorylation of Tyr(397), Tyr(407), Tyr(861), and Tyr(925) but not Tyr(576) or Tyr(577). To more specifically evaluate the kinetics of GRP/GRP-R upregulation, we wounded confluent cell monolayers. At t = 0 h GRP/GRP-R were not expressed, yet cells immediately began migrating into the gap created by the wound. GRP/GRP-R were first detected at approximately 2 h, and maximal levels were observed at approximately 6 h postwounding. The GRP-specific antagonist [d-Phe(6)]-labeled bombesin methyl ester had no effect on cell motility before GRP-R expression. In contrast, this agent increasingly attenuated cell motility with increasing GRP-R expression such that from t = 6 h onward no further cell migration into the gap was observed. Overall, these findings indicate the existence of GRP-independent and -dependent phases of tumor cell remodeling with the latter mediating colon cancer cell motility during remodeling via FAK.     

Expression-level dependent activation of recombinant human parathyroid hormone/parathyroid hormone-related peptide receptor: effect of human parathyroid hormone (1-34), (1-31), and (28-48)

A stable recombinant chinese hamster ovary (CHO) cell model system expressing the human type-1 receptor for parathyroid hormone and parathyroid hormone-related peptide (hPTH-R) was established for the analysis of human PTH (hPTH) variants. The cell lines showed receptor expression in the range from 10(5) to I.9 x 10(6) receptors per cell. The affinity of the receptors for hPTH-(1-34) was independent of the receptor number per cell (Kd approximately = 8 nmol/1). The induction of cAMP by hPTH-(1-34) is maximal in clones expressing >2x10(5) receptors per cell and Ca++ signals were maximal in cell lines expressing >1.4x10(6) receptors per cell. Second messenger specific inhibitors demonstrated that PTH-induced increases in intracellular cAMP and Ca++ are independent and Ca++ ions are derived from intracellular stores. The cAMP-specific receptor activator hPTH-(1-31) showed also an increase in intracellular Ca++. Even in cell lines expressing more than 10(6) receptors per cell the Ca++/PKC specific activator hPTH-(28-48) did not activate hPTH-Rs. Based on these results, synthesis of further derivatives of PTH is required to identify pathway-specific ligands for the type-1 hPTH-R.     

Overexpression and activation of the RON receptor tyrosine kinase in a panel of human colorectal carcinoma cell lines

RON is a receptor tyrosine kinase belonging to the MET proto-oncogene family. The purposes of this study are to determine the expression and activation of RON in a panel of human colon carcinoma cell lines. Western blotting showed that RON is barely detectable in normal and SV-40-transformed colon epithelial cells, but highly expressed and constitutively activated in several colon carcinoma cell lines including Colo201, HT-29, HCT116, and SW837. Moreover, a novel RON variant with a molecular mass of 160 kDa (RONDelta160) was identified from HT-29 cells. The cDNA encoding RONDelta160 has an in-frame deletion of 109 amino acids in the extracellular domain of the RON beta chain, which is caused by splicing out of two exons in the RON mRNA. No mutations were found in the kinase domain of the RON gene in five carcinoma cell lines screened. By expressing RON in colon epithelial cells, we found that RON activation increases cell motile-invasive activities and protects cells against apoptotic death. These data suggest that RON expression and activation are deregulated in colon carcinoma cell lines. By abnormal activation of RON, this receptor and its variant may regulate motile-invasive phenotypes of certain colon carcinoma cells in vivo.     

The case for gastrin-releasing peptide acting as a morphogen when it and its receptor are aberrantly expressed in cancer

Gastrin-releasing peptide (GRP) and its receptor (GRP-R) are frequently expressed by cancers of the gastrointestinal tract, breast, lung, and prostate. Most studies have found that GRP and its amphibian homologue bombesin act to increase tumor cell proliferation, leading to the hypothesis that this peptide hormone is a mitogen important for the growth of various cancers. Yet GRP/GRP-R co-expression in cancer promotes the development of a well-differentiated phenotype; while multiple studies suggest that the presence of these 2 proteins confer a survival advantage. Along with recent reports showing that GRP and its receptor critically regulate aspects of colon and lung organogenesis, we argue that these proteins do not function primarily as mitogens when aberrantly expressed in cancer. Rather, we postulate that GRP/GRP-R are onco-fetal antigens that function as morphogens, with their effect on tumor cell proliferation being a component property of their ability to regulate differentiation. Thus aberrant GRP/GRP-R expression in cancer recapitulates, albeit in a dysfunctional manner, their normal role in development.     

Contribution of gastrin-releasing peptide and its receptor to villus development in the murine and human gastrointestinal tract

Recent studies have shown that aberrantly expressed gastrin-releasing peptide (GRP) and its receptor (GRP-R) critically regulate tumor cell differentiation in colon cancers developing in humans and mice. This finding suggested that the ability of GRP/GRP-R to promote a well-differentiated phenotype in colon cancer might reflect a re-capitulation of a normal role in regulating intestinal organogenesis. To determine if this was the case, we compared and contrasted intestinal development in GRPR-/- mice with their wild type littermates. GRP/GRP-R co-expression in wild type mice was only observed in villous enterocytes between N-1 and N-12. During this time frame villous growth was completely attenuated in GRPR-/- mice. The contribution of GRP/GRP-R to villous growth was due to their act in increasing enterocyte proliferation prior to N-8 but increasing enterocyte size thereafter. From N-12 onwards, small intestinal villous growth in GRPR-/- mice resumed such that no difference in this structure could be detected at adulthood between mice of either genotype. We next studied GRP/GRP-R expression in human abortuses. These proteins were co-expressed by villous enterocytes only between weeks 14 and 20 post-conception, a time frame analogous to when they are expressed in the murine intestine. Thus, this study shows for the first time that GRP/GRP-R play a transient and non-critical role in intestinal development, yet provides a rationale for their re-appearance in colon cancer.     

Expression - Level Dependent Activation of Recombinant Human Parathyroid Hormone/Parathyroid Hormone-Related Peptide Receptor: Effect of Human Parathyroid Hormone (1-34), (1-31), and (28-48)

Abstract

A stable recombinant Chinese hamster ovary (CHO) cell model system expressing the human type-1 receptor for parathyroid hormone and parathyroid hormone-related peptide (hPTH-R) was established for the analysis of human PTH (hPTH) variants. The cell lines showed receptor expression in the range from 10⁵ to 1.9xl0⁶ receptors per cell. The affinity of the receptors for hPTH-(l-34) was independent of the receptor number per cell (K<j = 8 nmol/1). The induction of cAMP by hPTH-(l-34) is maximal in clones expressing >2xl0⁵ receptors per cell and Ca⁺⁺ signals were maximal in cell lines expressing >1.4xl0⁶ receptors per cell. Second messenger specific inhibitors demonstrated that PTH-induced increases in intracellular cAMP and Ca⁺⁺ are independent and Ca⁺⁺ ions are derived from intracellular stores. The cAMP-specific receptor activator hPTH-(l-31) showed also an increase in intracellular Ca⁺⁺. Even in cell lines expressing more than 10 receptors per cell the Ca⁺⁺/PKC specific activator hPTH-(28-48) did not activate hPTH-Rs. Based on these results, synthesis of further derivatives of PTH is required to identify pathway-specific ligands for the type-1 hPTH-R.     

Gastrin-releasing peptide induces itch-related responses through mast cell degranulation in mice

Gastrin-releasing peptide (GRP), secreted from the central terminals of primary afferents, is involved in the transmission of itch signals in the spinal dorsal horn. Although primary afferents containing GRP are distributed throughout the skin, the role of peripherally released GRP in the itch response is unknown. We investigated whether GRP acts on the skin to induce an itch response in mice. Intradermal injections of GRP(18-27) (1-300 nmol/site) elicited scratching. GRP(18-27)-induced scratching was inhibited by the μ-opioid receptor antagonist naltrexone hydrochloride, the BB(2) bombesin receptor antagonist RC-3095, the H(1) histamine receptor antagonists fexofenadine hydrochloride and chlorpheniramine maleate, and the PAR(2) proteinase-activated receptor antagonist FSLLRY-NH(2). Mast cell deficiency significantly, but not completely, reduced the GRP(18-27)-induced scratching. BB(2) bombesin receptors are present in mast cells in the skin, and intradermal injection of GRP(18-27), not only induced scratching, but also led to mast cell degranulation. GRP(18-27)-induced mast cell degranulation was inhibited by the BB(2) bombesin receptor antagonist RC-3095. These results suggest that peripherally released GRP can induce an itch response, at least partly, through activation of BB(2) receptors present in the mast cells, triggering their degradation and the release of histamine and the serine proteinase, tryptase.     

Substance P analogues function as bombesin receptor antagonists and inhibit small cell lung cancer clonal growth

Human small cell lung cancer (SCLC) produces and secretes BN/GRP (bombesin/gastrin releasing peptide). Because BN stimulates the growth of SCLC cells and these cells have receptors for BN-like peptides, it is important to define agents which disrupt this self-promoting autocrine growth cycle. Here, substance P analogues were evaluated as BN receptor antagonists using SCLC cell lines. (D-Arg¹, D-Pro², D-Trp^7,9, Leu¹¹) substance P [(APTTL)SP] was one of the more potent analogues tested in inhibiting BN-like peptide receptor binding with an IC₅₀ value of 1 μM. Micromolar concentrations of (APTTL)SP antagonized BN receptor mediated elevation of cytosolic Ca²⁺ levels and decreased the colony formation in soft agarose. These data suggest that SP analogues function as SCLC BN receptor antagonists and may be useful in disrupting the autocrine growth function of BN-like peptides.     

High affinity receptors for bombesin/GRP-like peptides on human small cell lung cancer

The binding of a radiolabeled bombesin analogue to human small cell lung cancer (SCLC) cell lines was investigated. (125I-Tyr4)bombesin bound with high affinity (Kd = 0.5 nM) to a single class of sites (2,000/cell) using SCLC line NCI-H446. Binding was reversible, saturable and specific. The pharmacology of binding was investigated using NCI-H466 and SCLC line NCI-H345. Bombesin and structurally related peptides, such as gastrin releasing peptide (GRP), but not other peptides, such as substance P or vasopressin, inhibited high affinity (125I-Tyr4)BN binding activity. Finally, the putative receptor, a 78,000 dalton polypeptide, was identified by purifying radiolabeled cell lysates on bombesin or GRP affinity resins and then displaying the bound polypeptides on sodium dodecylsulfate polyacrylamide gels. Because SCLC both produces bombesin/GRP-like peptides and contains high affinity receptors for these peptides, they may function as important autocrine regulatory factors for human SCLC.     

Biotinylation of a bombesin/gastrin-releasing peptide analogue for use as a receptor probe.

The development of a biotinylated bombesin/gastrin-releasing peptide (GRP) for use as a receptor probe is reported. The lysine13 of a GRP-27 was substituted by arginine and lysine was added to the amino terminus. Biotinylation of the N-terminal lysine was performed. The biotinylated peptide was purified by HPLC and characterized by mass spectral analysis. Binding studies with murine Swiss 3T3 fibroblasts, cells known to express bombesin/GRP receptors, yielded a dissociation curve for the biotinylated GRP-27 analogue (biotin-Lysyl[Asp12,Arg13]GRP-27) which was nearly identical to that of native GRP. Using studies of gastrin release from isolated canine G cells, equipotent functional activity of the biotinylated probe and unmodified GRP was demonstrated. Measurements of retained 125I-avidin confirmed that the biotin/avidin interaction could occur once the biotin-peptide complex was bound. Applicability of the probe was demonstrated with fluorescent microscopy using avidin-FITC on Swiss 3T3 fibroblasts. In conclusion, a novel biotinylated bombesin/GRP analogue has been developed which retains the functional characteristics of the native peptide and is a useful probe for receptor studies.     

Transfer of GRP94(Gp96)-associated peptides onto endosomal MHC class I molecules

GRP94 (gp96)-associated peptides can elicit cellular immune responses, an activity thought to reflect the presence of a cell surface receptor (CD91) on antigen-presenting cells that mediates GRP94 internalization and trafficking to an amenable site for peptide transfer to major histocompatibility complex class I molecules. We report that GRP94 internalized by receptor-mediated endocytosis is trafficked to a Rab5a, CD1 and transferrin-negative, Fc receptor and major histocompatibility complex class I-positive endocytic compartment. Receptor-internalized GRP94 did not access the endoplasmic reticulum of antigen-presenting cells. To identify the site of re-presentation of GRP94-associated peptides, kinetic analyses were performed utilizing GRP94-OVA (SIINFEKL) peptide complexes, with peptide re-presentation assayed with the K^b-SIINFEKL-specific MAb, 25-D1.16. Analyses of the kinetics of re-presentation of GRP94-associated peptides, under conditions in which de novo synthesis of major histocompatibility complex class I molecules was inhibited, identified a post-endoplasmic reticulum compartment, accessed by mature major histocompatibility complex class I, as the predominant site of GRP94-associated peptide exchange onto major histocompatibility complex class I.