A new approach using tissue alkaline phosphatase activity to identify early testicular cancer

Dabare, A.A.N.P.M.; Nouri, A. M. E.; Reynard, John; Killala, S.; Oliver, R.T.D.

doi:10.1046/j.1464-410x.1997.03526.x

Cited by 10 publications

(13 citation statements)

References 4 publications

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“…Similar steps were undertaken for phosphatase staining [6]. Briefly, slides were fixed in acetone and PBS + NaN 3 in the same way as immunostaining and the cellular phosphatase activity, i.e.…”

Section: Immunostaining and Phosphatase Stainingmentioning

confidence: 99%

“…In testicular malignancies, mainly seminomas, PLAP expression is elevated and can therefore serve as a biological marker [5,6]. In addition, the higher intensity of PLAP expression in seminoma compared with the more malignant and less differentiated teratoma has led to the suggestion that PLAP might be considered as a marker of differentiation [7][8][9][10].…”

Section: Introductionmentioning

confidence: 99%

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Profile of Placental Alkaline Phosphatase Expression in Human Malignancies: Effect of Tumour Cell Activation on Alkaline Phosphatase Expression

Dabare¹,

Nouri²,

Cannell

et al. 1999

Urol Int

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Cellular alkaline phosphatases (ALP) are increasingly recognised as important markers for monitoring tumour cell behaviour in human malignancies. Colorimetric, flow-cytometric, and immunocytochemical assays were employed to assess the influence of activation on expression of cellular ALP in human tumour cell lines. The results showed the following: (1) Testis tumour biopsies (16/16) unlike bladder (0/14) and head and neck (0/16) tumours showed positive staining for ALP, particularly the placental type, i.e. PLAP, although this was not always present on all the cells of non-seminoma biopsies. (2) The intensity of ALP expression differed widely in tumour cell lines. Based on biochemical analysis, the profile of ALP fell into two categories: (a) low expressing (MW 70 kD, placental type ALP) like Hep2 and KB lines, and (b) those expressing both low and high molecular (MW 95 kD) bands like testis lines Tera II and Ep2102. In all cases treatment of tumour cell lysates with heat prior to biochemical analysis showed the disappearance of the higher and sharpening of the lower molecular weight ALP band. (3) Exposure of tumour cells to epidermal growth factor (EGF) expressing EGF receptor led to a decreased ALP expression by as much as 54% as assessed by biochemical or flow-cytometric techniques. These data demonstrated that testis tumour tissues and cell lines expressed ALP which were different from others. The data also showed that exposure of tumour cell lines expressing EGFr to EGF resulted in suppression of ALP expression. These observations are consistent with the notion that EGFr and PLAP expression may be taken as a marker of proliferation and differentiation in human malignancies, respectively.

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Section: Immunostaining and Phosphatase Stainingmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Profile of Placental Alkaline Phosphatase Expression in Human Malignancies: Effect of Tumour Cell Activation on Alkaline Phosphatase Expression

Dabare¹,

Nouri²,

Cannell

et al. 1999

Urol Int

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“…Previous reports from Dabare et al [11] showed the unique two-band ALP profile in testis tumours using Western-blot analysis as well as the MEDC method. In the present study using the more robust and reliable HPLC system, the presence of a two-band profile in testis cancer and testis cell lines was confirmed.…”

Section: Discussionmentioning

confidence: 99%

“…Testis cancers have been of particular interest as PLAP is highly expressed in well-differentiated testis tumour, i.e. seminoma, and less so in more poorly differentiated teratomas [11]. This has led to the initial enthusiasm to use PLAP sera levels as a marker for testis cancers.…”

Section: Discussionmentioning

confidence: 99%

“…The colorimetric assay was first reported by Dabare et al [11]. This technique was prepared as follows: immobulin paper used for dot-blot technique was loaded with the test sample and left in solution of one tablet of ALP substrate (5-bromo-4-chloro-3-indolyl phosphatase/interblue tetrazolium tube) in 10 ml distilled water for more than 10 min.…”

Section: Medc Assaymentioning

confidence: 99%

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Standardization and Potential Use of HPLC for Detection of Cellular Placental Alkaline Phosphatase Using Established Tumour Cell Lines and Fresh Tumour Biopsies

Torabi‐Pour¹,

Nouri²,

Chineguwndo³

et al. 1999

Urol Int

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Placental alkaline phosphatase (PLAP) is one of the cellular phosphatases (ALP) expressed in patients with testis cancers, particularly in seminomas. Using various techniques including Western blot and high-performance liquid chromatography (HPLC) systems and ATC2, a newly developed specific anti-PLAP monoclonal antibody (Mab), the presence of active form of PLAP in lysates prepared from testis tumour fragment and tumour cell lines, was studied. This was carried out following isolation of PLAP from biological samples using CNBr Sepharose-conjugated ATC2 beads. The results showed that: (1) The target for the newly developed Mab ATC2 was PLAP. (2) The ATC2-conjugated bead system was an efficient method for isolating pure PLAP. (3) Diethylamine (DEA), in contrast to urea and glycine, was the most efficient for separation of PLAP from ATC2-conjugated beads, as the isolated molecule did not lose any phosphatase activity and there was very little uncoupling of the ATC2 Mab from the beads. (4) ATC2-conjugated CNBr beads could pick up PLAP from a solution containing standard PLAP and lysates prepared from tumour cell lines or testis tissue fragments positive for the PLAP. (5) HPLC profile of testis tumour lines and testis tumours showed two distinct peaks with ALP activity, one at retention time 7– 8 min (corresponding to 95 kDa molecule) and one at 12–13 min corresponding to 70 kDa molecule). These data demonstrated the potential use of various biochemical methods in combination with HPLC for isolation of the fully functional molecules with ALP activity from different samples including lysates prepared from patients with testis cancer. The nature of ALP activity at 95 kDa is being investigated as no such molecule has been reported previously. These techniques might have an important implication for an early detection of germ cell tumours, particularly in patients with equivocal ultrasound.

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The combined use of high performance liquid chromatography and immuno‐biochemical techniques for protein isolation: a new approach for identification of an individual protein from a pool of proteins

Torabi‐Pour

Nouri

Perrett

et al. 2000

Biomedical Chromatography

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HPLC was used in combination with immuno-bead separation technique for identification of an individual protein from a pool of proteins. This was carried out using an in-house monoclonal antibody (ATC2) specific for placental alkaline phosphatase (PLAP) as a primary antibody for conjugation to CNBr beads. The phosphatase activity (ALP) of PLAP was measured by colorimetric assay (MEDC). The data from this study has so far indicated that: 1. HPLC analysis of molecules following isolation with ATC2-conjugated beads showed high degree of purity. This could be achieved using protein mixtures prepared from lysates of tumour cell lines or tumour fragments. 2. HPLC-isolated PLAP maintained phosphatase activity. 3. Out of the four dissociation reagents used, diethyl amine (DEA) was found to be the best reagent for dissociation of antigen, ie PLAP, but not mAb from CNBr beads. 4. The profile of ALP activity was different for samples prepared from testis and kidney fragments, both in terms of the HPLC peak profile as well as the sensitivity. These data confirmed that the immuno-bead separation technique in conjunction with HPLC were powerful tools for identifying an individual protein from a pool of proteins. These approaches are being used for the identification of PLAP molecules, as a tumour marker in patients suspected of testicular malignancies with equivocal ultrasound.

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A new approach using tissue alkaline phosphatase activity to identify early testicular cancer

Cited by 10 publications

References 4 publications

Profile of Placental Alkaline Phosphatase Expression in Human Malignancies: Effect of Tumour Cell Activation on Alkaline Phosphatase Expression

Profile of Placental Alkaline Phosphatase Expression in Human Malignancies: Effect of Tumour Cell Activation on Alkaline Phosphatase Expression

Standardization and Potential Use of HPLC for Detection of Cellular Placental Alkaline Phosphatase Using Established Tumour Cell Lines and Fresh Tumour Biopsies

The combined use of high performance liquid chromatography and immuno‐biochemical techniques for protein isolation: a new approach for identification of an individual protein from a pool of proteins

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