Quantitative immunofluorescence, anti-ras p21 antibody specificity, and cellular oncoprotein levels

Jones, Philip L.; O’Hare, Christopher; Bass, Rebecca A.; Rao, Jian Yu; Hemstreet, George P.; Hurst, Robert E.

doi:10.1016/0006-291x(90)92046-3

Cited by 13 publications

(6 citation statements)

References 17 publications

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“…The elevation in p185 in nearly 70% of all tumors and 82% of high-grade tumors exceeds the 36% found by Northern analysis and 21% found by immunohistochemistry (31). These apparent discrepancies are understandable; the normalization of mRNA levels to actin mRNA may depress the ratio if actin biosynthesis is increased in transformed cells (26), and immunofluorescence can be more accurately quantitative and sensitive than immunohistochemistry (22,32).…”

Section: Resultsmentioning

confidence: 94%

“…A corresponding negative control slide omitting primary antibodies was also prepared. All reagent concentrations were optimized to achieve saturation of cellular binding sites (22,24). Each batch of reagent was individually titrated to determine the appropriate concentration that produced saturation and, hence, proportionality between immunofluorescence and the target biomolecule.…”

Section: Methodsmentioning

confidence: 99%

“…The touch preparation slides were dewaxed with 95% (vol/vol) ethanol for 15 min, followed by 10 dips each in 70% and 50% ethanol and soaking in acetone for 5 min. The slides were triple-labeled for DNA by using 15 ,uM Hoechst 33258 (20), for p185 by using the TAl antibody (21) directly conjugated to Texas red, and for p300 by using the M344 mouse monoclonal antibody (17) and a three-stage sequence using biotin-conjugated goat antibody to mouse IgGl as secondary antibody and fluorescein-labeled avidin (22). With 14 of the tumors and 5 of the controls a second triple-labeled slide was prepared, labeling for DNA as above, for EGFR by using AB-1 mouse monoclonal antibody (Oncogene Science) and the same visualization system as was used with M344, and for G-actin by using DNase I (Molecular Probes) directly conjugated to Texas red (23).…”

Section: Methodsmentioning

confidence: 99%

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Alterations in phenotypic biochemical markers in bladder epithelium during tumorigenesis.

Rao

Hemstreet

Hurst

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

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Phenotypic biochemical markers of oncogenesis and differentiation were mapped in bladder biopsies to investigate changes that occur in bladder tumorigenesis and to identify markers for increased bladder cancer risk. Touch preparations from biopsy specimens from 30 patients were obtained from tumors, the adjacent bladder epithelium, and random distant bladder epithelium. Markers, including DNA ploidy, epidermal growth factor receptor (EGFR), and oncoproteins, were quantified in individual cells by using quantitative fluorescence image analysis. Cluster analysis revealed the markers fell into three independent groups: (0) G-actin and EGFR; (it) ploidy, cytology, and p185 (HER-2/neu oncoprotein) (ERBB2); and (iil) p300, a low-grade tumor antigen. Each marker displayed a gradient of abnormality from distant field to adjacent field to tumor. Different patterns for each marker suggested a developmental sequence of bladder cancer oncogenesis; G-actin was altered in 58% of distant biopsies (vs. 0/6 normals, P < 0.001), ploidy and cytology were altered in <20% of distant fields and =80% of tumors, and the other markers were intermediate. Patterns of EGFR and p185 suggest lowand high-grade tracks diverge early (P < 0.05 by MannWhitney U test for EGFR and ANOVA for p185). In conclusion, this study shows that a sequence of phenotypic changes accompanies development and progression of bladder cancers. Biochemical alterations in cells of the bladder field are often detectable before abnormal pathology, and markers previously thought to be limited to tumors were found in the field. The hierarchy of expression may be useful in identifying high-risk patients, assessing completeness of response to therapy, and monitoring and predicting recurrence.Decreasing future deaths from bladder cancer will require a strategy of prevention, based in part upon identification of individuals at risk for invasive metastatic disease well before it becomes symptomatic. Recognition that clinical cancer is the end-point of the underlying disease of carcinogenesis (1) suggests that appropriate intervention with individuals determined to be at risk could prevent development of lifethreatening disease.The concept of "field cancerization" or "field disease," introduced in 1953 (2), suggests that the whole field of tissue is exposed to carcinogen and is at increased risk for developing cancers, even though individual lesions are of clonal origin. The field disease theory as applied to bladder cancer has provided a central organizing theory for understanding these multifocal, frequently recurrent tumors (3-5). The normal bladder, or any other solid organ, represents a complex ecosystem ofinteracting epithelial and stromal cells, and years are required for the progressive subversion of growth and differentiation controls (6-8) to result in eventual emergence of cells capable of at least partial autonomousThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" i...

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Section: Resultsmentioning

confidence: 94%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Alterations in phenotypic biochemical markers in bladder epithelium during tumorigenesis.

Rao

Hemstreet

Hurst

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

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“…European investigators evaluated a battery of antibodies, and although the sensi tivity compared to conventional cytology was increased, the specificity was poor ( Immunocytochemistry is a semiquantitative test and is usually reported as 'percent of cells positive'. This param eter is arbitrary, in that the fraction of cells positive is dependent on the antibody concentration and can be var ied from 0 to 100% by varying the antibody concentration [6], On the other hand, biomarkers may be helpful in con ditions that are poorly distinguished by cytology by de tecting risk for bladder cancer in screening of high-risk groups, monitoring chemoprevention therapy and indi vidual risk assessment. The earlier the marker becomes positive the lower its specificity for predicting progression at each stage of carcinogenesis.…”

Section: Biomarkers As Adjunct To Urinary Cytologymentioning

confidence: 99%

Diagnosis and Treatment of Superficial Transitional Cell Carcinoma of the Bladder: Facts and Perspectives

Kh¹

1997

Eur Urol

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“…These earlier ras protein studies often used one of the pan-ras antibodies, Rap5 or Y13-259, concluding that this antibody measured overexpression of H-ras protein or of all ras proteins (Jansson et al, 1990). The Rap5 antibody has now been identified as showing non-specific binding to ras proteins so that it cannot be used in most instances to detect the overexpression of either a particular ras protein nor to confirm the presence of all 3 ras proteins (Jones et al, 1990). The other well-known pan-ras antibody, Y13-259, has given extremely different results in different laboratories.…”

Section: H-and K-ras Protein Levels In Colon Cancermentioning

confidence: 97%

N-ras protein: Frequent quantitative and qualitative changes occur in human colorectal carcinomas

et al. 1997

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Point mutation and overexpression are recognized mechanisms for ras activation in malignancy. However, little information is available on overexpression of N-ras protein compared with H-or K-ras proteins, as N-ras-specific antibodies have only recently become available. For comparative analyses of ras protein levels, we have probed Western blots of extracts from 9 normal human tissues and 55 pairs of colorectal carcinoma and matched control mucosa, using monoclonal antibodies (MAbs) specific for H-, K-or N-ras proteins. On multi-tissue blots, N-ras protein was more highly expressed in colon than in the other human tissues analyzed, suggesting a role for N-ras in colorectal function. N-ras protein levels in multiple independent extracts of normal colon mucosa were consistently higher than either K-ras protein or H-ras protein levels. In 69% of colon carcino-mas, N-ras protein levels were increased an average of 4.8-fold over normal mucosa. Overexpression of K-ras protein was also observed in colon cancers but less frequently (13% of cases) than N-ras protein. H-ras protein levels were too low for comparative studies. Alterations in N-ras protein mobility, possibly reflecting increased post-translational processing , were also detected in 42% of colon carcinomas. N-ras protein, typically present as a single 23 kDa band in normal mucosa, was expressed in some cancers as a 22 kDa band or as multiple bands of 20-23 kDa. Sequencing of N-ras DNA from 6 carcinomas with these variations in protein mobility did not reveal mutations in codons 12, 13, 59 or 61. Thus, frequent quantitative and qualitative changes in N-ras protein expression, which do not appear to correlate with the presence of typical N-ras point mutations, result in abnormal N-ras protein patterns in human colorectal carcinomas. Int.

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Quantitative immunofluorescence, anti-ras p21 antibody specificity, and cellular oncoprotein levels

Cited by 13 publications

References 17 publications

Alterations in phenotypic biochemical markers in bladder epithelium during tumorigenesis.

Alterations in phenotypic biochemical markers in bladder epithelium during tumorigenesis.

Diagnosis and Treatment of Superficial Transitional Cell Carcinoma of the Bladder: Facts and Perspectives

N-ras protein: Frequent quantitative and qualitative changes occur in human colorectal carcinomas

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