H.-J. Leisinger

Clinical evaluation of a method for detecting superficial transitional cell carcinoma of the bladder by light-induced fluorescence of protoporphyrin IX following topical application of 5-aminolevulinic acid: Preliminary results

In bladder cancer, conventional white light endoscopic examination of the bladder does not provide adequate information about the presence of “flat” urothelial lesions such as carcinoma in situ. In the present investigation, we examine a new technique for the photodetection of such lesions by the imaging of protoporphyrin IX (PpIX) fluorescence following topical application of 5‐aminolevulinic acid (ALA).

Enzymatic profiling of human kallikrein 14 using phage-display substrate technology

Abstract The human KLK14 gene is one of the newly identified serine protease genes belonging to the human kallikrein family, which contains 15 members. KLK14, like all other members of the human kallikrein family, is predicted to encode for a secreted serine protease already found in various biological fluids. This new kallikrein is mainly expressed in prostate and endocrine tissues, but its function is still unknown. Recent studies have demonstrated that KLK14 gene expression is up-regulated in prostate and breast cancer tissues, and that higher expression levels correlate with more aggressive tumors. In this work, we used phage-display substrate technology to study the substrate specificity of hK14. A phage-displayed random pentapeptide library with exhaustive diversity was screened with purified recombinant hK14. Highly specific and sensitive substrates were selected from the library. We show that hK14 has dual activity, trypsin- and chymotrypsin-like, with a preference for cleavage after arginine residues. A SwissProt database search with selected sequences identified six potential human protein substrates for hK14. Two of them, laminin α-5 and collagen IV, which are major components of the extracellular matrix, have been demonstrated to be hydrolyzed efficiently by hK14.

Fluorescence Cystoscopy in the Management of Bladder Cancer: A Help for the Urologist!

As a disease characterized by a nature polymorphic and fluctuant in its evolution, superficial transitional cell carcinoma of the bladder remains a perpetual therapeutic challenge, and raises a great interest in the development of new diagnostic and surveillance techniques. This paper reviews 10 years of experience of fluorescence cystoscopy, a simple technique perfectly adapted to the current endoscopic equipment. Its principle is to enhance the visual contrast between benign and malignant cells. Three photosensitizing agents are available, two prodrugs: δ-aminolevulinic acid or hexaminolevulinate, and a natural substance: hypericin. With a detection rate of over 90% for carcinoma in situ and a real potential for detecting small tumors overlooked by standard cystoscopy, fluorescence cystoscopy may be clearly recommended in clinical practice. This technique favors a standardization of superficial bladder cancer endoscopic management and is susceptible to have a real impact on the disease recurrence and progression rate.

Photodynamic therapy in superficial bladder cancer: past, present and future

Abstract For many reasons, such as toxicity and lack of selectivity of photosensitisers, as well as complexity of technical procedures and inconstant therapeutic results, photodynamic therapy of highly recurrent superficial bladder cancer never gained wide acceptance in the urological community. However, the 25 years of experience combined with the recent discovery of new photosensitisers, such as protoporphyrin IX (PpIX) induced by 5-aminolevulinic acid (ALA) or ALA-derivatives or hypericin open new, very interesting perspectives in this therapeutic field.

Evaluation of the CFP-substrate-YFP system for protease studies: advantages and limitations

A protease can be defined as an enzyme capable of hydrolyzing peptide bonds. Thus, characterization of a protease involves identification of target peptide sequences, measurement of activities toward these sequences, and determination of kinetic parameters. Biological protease substrates based on fluorescent protein pairs, which allow for use of fluorescence resonance energy transfer (FRET), have been recently developed for in vivo protease activity detection and represent a very interesting alternative to chemical substrates for in vitro protease characterization. Here, we analyze a FRET system consisting of cyan and yellow fluorescent proteins (CFP and YFP, respectively), which are fused by a peptide linker serving as protease substrate. Conditions for CFP-YFP fusion protein production in Escherichia coli and purification of proteins were optimized. FRET between CFP and YFP was found to be optimum at a pH between 5.5 and 10.0, at low concentrations of salt and a temperature superior to 25 degrees C. For efficient FRET to occur, the peptide linker between CFP and YFP can measure up to 25 amino acids. The CFP-substrate-YFP system demonstrated a high degree of resistance to nonspecific proteolysis, making it suitable for enzyme kinetic analysis. As with chemical substrates, substrate specificity of CFP-substrate-YFP proteins was tested towards different proteases and kcat/Km values were calculated.

Mutant recombinant serpins as highly specific inhibitors of human kallikrein 14

The reactive center loop (RCL) of serpins plays an essential role in the inhibition mechanism acting as a substrate for their target proteases. Changes within the RCL sequence modulate the specificity and reactivity of the serpin molecule. Recently, we reported the construction of α1‐antichymotrypsin (ACT) variants with high specificity towards human kallikrein 2 (hK2) [Cloutier SM, Kündig C, Felber LM, Fattah OM, Chagas JR, Gygi CM, Jichlinski P, Leisinger HJ & Deperthes D (2004) Eur J Biochem271, 607–613] by changing amino acids surrounding the scissile bond of the RCL and obtained specific inhibitors towards hK2. Based on this approach, we developed highly specific recombinant inhibitors of human kallikrein 14 (hK14), a protease correlated with increased aggressiveness of prostate and breast cancers. In addition to the RCL permutation with hK14 phage display‐selected substrates E8 (LQRAI) and G9 (TVDYA) [Felber LM, Borgoño CA, Cloutier SM, Kündig C, Kishi T, Chagas JR, Jichlinski P, Gygi CM, Leisinger HJ, Diamandis EP & Deperthes D (2005) Biol Chem386, 291–298], we studied the importance of the scaffold, serpins α1‐antitrypsin (AAT) or ACT, to confer inhibitory specificity. All four resulting serpin variants ACTE8, ACTG9, AATE8 and AATG9 showed hK14 inhibitory activity and were able to form covalent complex with hK14. ACT inhibitors formed more stable complexes with hK14 than AAT variants. Whereas E8‐based inhibitors demonstrated a rather relaxed specificity reacting with various proteases with trypsin‐like activity including several human kallikreins, the two serpins variants containing the G9 sequence showed a very high selectivity for hK14. Such specific inhibitors might prove useful to elucidate the biological role of hK14 and/or its implication in cancer.

Clinical assessment of fluorescence cystoscopy during transurethral bladder resection in superficial bladder cancer

The prognosis of superficial bladder cancer in terms of recurrence and disease progression is related to bladder tumor multiplicity and the presence of concomitant “plane” tumors such as high-grade dysplasia and carcinoma in situ. This study in 33 patients aimed to demonstrate the role of fluorescence cystoscopy in transurethral resection of superficial bladder cancer. The method is based on the detection of protoporphyrin-IX-induced fluorescence in urothelial cancer cells by topical administration of 5-aminolevulinic acid. The sensitivity and the specificity of this procedure on apparently normal mucosa in superficial bladder cancer are estimated to be 82.9% and 81.3%, respectively. Thus, fluorescence cytoscopy is a simple and reliable method for mapping the bladder mucosa, especially in the case of multifocal bladder disease, and it facilitates the screening of occult dysplasia.

Profiling of proteolytic activities secreted by cancer cells using phage display substrate technology.

Although the cellular steps required for metastasis are similar for all cancer cells, proteases involved in this process and their expression levels vary greatly between different cancer types. Thus, the identification of these proteolytic activities represents a crucial issue in the understanding of cancer development. Until now, phage display substrate technology has been successfully employed for the characterization of purified proteases but was never used with a mix of proteases. In the present work, we report an easy protocol to identify multiple proteolytic activities secreted by cancer cells. We selected substrates from a phage display library of high diversity using secreted media of three established prostate cancer cell lines (DU-145, LNCaP and PC-3) with variable degrees of invasive capability. Some of these selected peptide substrates were hydrolyzed by the secreted proteins of all three prostatic cancer cell lines, demonstrating similarities in their proteolytic activities. On the other hand, a few substrates were cancer cell specific, indicating differences in the phenotypes of protease expression in prostate cancer. This work reports for the first time the selection of substrates from a mix of proteases using phage display technology and opens a new avenue for the direct identification of proteolytic activities for tumor extracts.

Peptabody-EGF: A novel apoptosis inducer targeting ErbB1 receptor overexpressing cancer cells

The epidermal growth factor receptor (EGFR) plays a central role in cell life by controlling processes such as growth or proliferation. This receptor is commonly overexpressed in a number of epithelial malignancies and its upregulation is often associated with an aggressive phenotype of the tumor. Thus, targeting of EGFR represents a very promising challenge in oncology, and antibodies raised against this receptor have been investigated as potential antitumor agents. Various putative mechanisms of action were proposed for such antibodies, including decreased proliferation, induction of apoptosis, stimulation of the immunological response against targeted cancer cells or combinations thereof. We report here the development of an alternative high affinity molecule that is directed against EGFR. Production of this pentameric protein, named peptabody‐EGF, includes expression in a bacterial expression system and subsequent refolding and multimerization of peptabody monomers. The protein complex contains 5 human EGF ligand domains, which confer specific binding towards the extracellular portion of EGFR. Receptor binding of the peptabody‐EGF had a strong antiproliferative effect on different cancer cell lines overexpressing EGFR. However, cells expressing constitutive levels of the target receptor were barely affected. Peptabody‐EGF treated cancer cells exhibited typical characteristics of apoptosis, which was found to be induced within 30 min after the addition of the peptabody‐EGF. In vitro experiments demonstrated a significantly higher binding activity for peptabody‐EGF than for the therapeutic monoclonal EGFR antibody Mab‐425. Furthermore, the antitumor action provoked by the peptabody‐EGF was greatly superior than antibody mediated effects when tested on EGFR overexpressing cancer cell lines. These findings suggest a potential application of this high affinity molecule as a novel tool for anti‐EGFR therapy.

On the influence of the instillation time on the results of HAL (Hexvix®) fluorescence detection of superficial bladder cancer

Hexyl aminolevulinate (HAL) fluorescence cystoscopy is being investigated as a new diagnostic tool for the detection of flat urothelial malignancies in bladder cancers. However, the influence of the bladder instillation time on the performance of this detection modality has not been addressed up to now. We report our initial experience comparing different instillation schedules of HAL cystoscopy in the diagnosis of superficial bladder cancer. A total of 718 fluorescent positive (433) and fluorescence negative (285) biopsies have been taken in the bladder of 143 patients using the Storz D-light fluorescence imaging system (Karl Storz, Tuttlingen, Germany) which allows both white and blue light (380-450 nm) bladder wall inspection. Following hospitalisation, 50 ml of HAL (8mM) phosphate buffer solution was instilled into the bladder of patients during one hour (1 hour protocol involving 57 patients), or during two hours followed by a two hours resting time after removal of the solution (2+2 hours protocol involving 86 patients). Both instillation subgroups were homogeneous in terms of proportion of high risk disease, previous BCG treatment and/or recurrent disease. This study indicates that the instillation duration does not influence the results of HAL (Hexvix) fluorescence cystoscopy in our conditions. Compared to the standard use of ALA, HAL (Hexvix) fluorescence cystoscopy allows a significant reduction of the instillation time (to less than one hour) without prejudicing the efficacy of the method, what represents a real advantage in daily clinical practice.