Detection of genes expressed in primary colon cancers by in situ hybridisation: overexpression of RACK 1

Aims: The isolation of various genes that are expressed in a region specific manner is considered useful for research in molecular pathology. In situ hybridisation (ISH) was used in a screening procedure to isolate these genes efficiently, using colon cancer as a model.

Methods: Suppression subtractive hybridisation (SSH) between colon cancer tissue samples and corresponding non-cancerous tissues was performed. Genes showing high expression in the cancers were selected using macro-DNA array analysis. As a final screening procedure, conventional ISH was performed to isolate genes expressed specifically in colon cancers.

Results: Sixty nine clones were selected by SSH and macro-DNA array analyses. These clones were then analysed by ISH to examine their expression patterns. ISH screening revealed that all the clones screened showed more intense signals in colon cancers than in non-cancerous tissues. Among them, RACK 1, which is a protein kinase C receptor and a homologue of the G protein β subunit, was expressed intensely in colon cancer cells. RACK 1 expression was evaluated in multiple samples by ISH, and the results confirmed that RACK 1 was universally overexpressed in cells of all 11 colon cancers examined.

Conclusions: Many genes, including RACK 1, expressed in colon cancer cells can be isolated efficiently by this method, and their precise expression pattern can be evaluated. These results indicate that ISH is an excellent technique for systemic screening of genes expressed in a region specific manner.

     

RACK1高表达促进人结肠癌细胞的生长和增殖

目的：研究RACK1高表达对人结肠癌细胞增殖能力的影响。方法：采用脂质体转染术分别建立 RACK1表达下调的SW620细胞系、RACK1表达上调的SW480细胞系以及对照细胞系;采用CCK-8细胞增殖测定、软琼脂集落形成实验、EdU掺入实验以及流式细胞术检测RACK1表达改变对 SW620和SW480细胞增殖的影响;采用Western blot分析RACK1表达改变对G1/S期限制点调控蛋白Cyclin D1和p27蛋白表达的影响。结果：下调RACK1的表达抑制SW620细胞的生长、软琼脂集落形成能力,降低EdU标记的S期细胞数目,阻滞细胞周期于G1/S期;而上调RACK1的表达增强 SW480细胞的生长、软琼脂集落形成能力,增加EdU标记的S期细胞数目,促进细胞周期G1/S期进程。结论：RACK1高表达促进人结肠癌细胞的生长和增殖。     

Detection of Campylobacter pylori in stomach tissue by DNA in situ hybridisation.

A non-radioactive DNA in situ hybridisation (DISH) protocol was developed. It requires the use of biotinylated Campylobacter pylori DNA as the probe to detect C pylori DNA in routinely embedded stomach biopsy specimens. In sequential tissue samples from a 58 year old woman with recurrent chronic active gastritis the C pylori probe hybridised with bacteria whenever they were histologically visible. When no bacteria were visible histologically, hybridisation was negative with one exception. In a single biopsy specimen without visible C pylori, hybridisation occurred with the surface of the antrum epithelium, while control hybridisation with a heterologous probe remained negative. From a parallel biopsy specimen taken at the same time the C pylori culture was positive. It is concluded that DISH, although more laborious than routine staining techniques, may be more sensitive in that it detects bacteria very easily, even when sections are not counterstained or when they are present in low numbers, and that bacteria which do hybridise are unequivocally identified as C pylori and not Campylobacter-like organisms.     

Detection of B19 parvovirus in human fetal tissues by in situ hybridisation.

Evidence of B19 parvovirus infection was sought by in situ hybridisation with biotinylated probes in 65 tissue samples from 32 pregnancies (fetuses, products of conception and/or placentas). Twenty-seven samples were reactive and the results were confirmed by other methods for B19 virus detection in 22 cases. The other methods used were in situ hybridisation with 3H and 35S labelled probes; dot-blot hybridisation with biotin and 32P labelled probes; polymerase chain reaction assay; negative stain and thin section electron microscopy; and radioimmunoassay for B19 antigen. The five false positive results by in situ hybridisation with biotinylated probes were considered to be due to non-specific biotin capture and were more frequent with unfixed samples than with formalin fixed material. It was concluded that while biotinylated probes offered advantages over radioactive probes for detecting B19 DNA by in situ hybridisation, positive findings should be confirmed by other methods.     

Detection of human papillomavirus DNA in urinary bladder carcinoma by in situ hybridisation.

AIMS: To investigate the sensitivity of an in situ hybridisation system to detect human papillomavirus (HPV) infection in transitional cell bladder cancer and to evaluate the advantages of analysing multiple biopsies; to examine the correlation between HPV tumour infection detected by in situ hybridisation and the presence of serum anti-HPV antibodies detected by enzyme linked immunosorbent assay (ELISA); and to relate the presence of viral infection to grade, stage, and follow up in cases of bladder cancer. METHODS: The in situ hybridisation technique was used with broad spectrum and type specific (6/11, 16/18, 31/33/35) probes against HPV DNA in formalin fixed, paraffin embedded tissues from 43 cases of bladder cancer. The results were analysed for the presence and type of papillomavirus and correlated with clinicopathological variables. RESULTS: The presence of HPV DNA was identified by the in situ hybridisation technique in 17 of 43 cases of bladder cancer; 12 of these were serum antibody positive and 10 had had multiple biopsies. Fifteen of the cases that were negative for HPV DNA by in situ hybridisation had positive serum serology when tested by ELISA. In 14 cases, the HPV was either types 16/18 or types 31/33/35, both of which carry high oncogenic risk. The stage (p < 0.05) and grade (NS) of the tumour and the outcome on follow up (p < 0.05) were correlated with the presence of HPV infection. CONCLUSIONS: ELISA is not useful in identifying patients with HPV positive bladder cancer, but the use of several probes and multiple biopsies increases the detection rate of HPV in neoplastic tissues. The association between tumour virus infection and high grade/high stage tumours and worse outcome suggests that HPV infection of neoplastic tissue has a negative effect on the behaviour and evolution of transitional cell bladder carcinoma.     

Detection of TEM beta-lactamase genes by non-isotopic spot hybridisation

A 998 bp fragment of plasmid pBR322, comprising part of the TEM-1-lactamase gene, was labelled with biotin-11-dUTP for use as a DNA probe in a rapid non-isotopic spot hybridisation test. Diluted broth cultures of bacteria producing different-lactamases were filtered onto nitrocellulose and lysed in situ. Following pre-hybridisation treatment with proteinase K, hybridisation with the labelled probe was demonstrated using a commercially available strep tavidine/poly alkaline phosphatase-based detection system. The probe was highly specific, reacting only with strains producing either the TEM-1 or structurally similar TEM-2 enzyme. An inoculum of 3–4 × 10⁶ cells gave optimum positive discrimination. When 90 recent ampicillin-resistant strains ofEscherichia coli isolated from patients with urinary tract infections were screened using the system, 72% gave a positive hybridisation signal.     

Detection of human papillomavirus infection by non-isotopic in situ hybridisation in condylomatous and CIN lesions.

AIMS--To study the value of non-isotopic in situ hybridisation (NISH) in detecting human papillomavirus (HPV) infection in female genital lesions positive for the virus by conventional histology but negative by filter DNA hybridisation. METHODS--Forty three cases, which showed the histological hallmarks of the HPV infection but produced negative results in filter dot blot hybridisation tests (Vira Pap and Vira Type kits), were identified in the course of an investigation of 304 vaginal, vulvar, and cervical samples from 267 patients. These cases were studied by NISH for the presence of HPV infection. RESULTS--In 28 (65%) of the cases NISH gave a positive hybridisation signal. In 26 cases (96%) the signal was diffuse, and in two (4%) punctate or diffuse, representing episomal and episomal or integrated HPV DNA, respectively. In most cases only a few HPV positive cells were discernible. CONCLUSION--NISH is a more sensitive technique than dot blot hybridisation, detecting HPV infection in most cases which show histological HPV atypia but which remain negative in filter DNA hybridisation. Thus NISH is useful as an additional technique to verify the presence of HPV in lesions which remain negative in filter hybridisation tests.     

Trisomy 10qter confirmed by in situ hybridisation.

We report a boy with multiple congenital anomalies compatible with trisomy for the distal region of the long arm of chromosome 10 and a male karyotype with one 18p+. In situ hybridisation with a cDNA for ornithine aminotransferase (OAT), whose locus maps to 10q26, confirmed the clinical suspicion of distal trisomy 10q. Subterminal localisation of the labelling signals on chromosome 10 and on the der(18) indicated the localisation of the OAT locus in the proximal part of 10q26. Two clusters of labelling signals were also found on the pericentromeric and proximal portion of the X chromosome short arm, thus confirming the presence in this region of two non-adjacent OAT pseudogenes. The phenotypic similarities of this patient to previously reported cases provide further support for the delineation of trisomy 10qter as a specific, clinically recognisable syndrome.     

In situ hybridisation in perspective.

In the introduction to this review two questions were posed: is the technology associated with ISH ready for general use, and will the method become an important investigative tool? With the exception of the demonstration of some single and low copy sequences, non-radioactive ISH is now sufficiently developed and simplified to make it a routine technique. It is also clear that ISH will continue to have an important research role. In diagnostic pathology the technique is already providing valuable information and the present decade should see the development of many more diagnostic applications.     

Detection of immunoglobulin light chain mRNA by in situ hybridisation using biotinylated tyramine signal amplification.

A highly sensitive method for the light microscopic in situ hybridisation of immunoglobulin light chain mRNA in formalin fixed, paraffin wax embedded sections is reported. This method is based on signal amplification using horseradish peroxidase catalysed deposition of biotinylated tyramine at the sites of hybridisation. kappa and lambda light chain immunoglobulin mRNA in situ hybridisation was performed with fluorescein isothiocyanate conjugated oligonucleotide probe cocktails. The hybridisation signal was detected using a biotinylated tyramine signal amplification procedure with streptavidinbiotin-horseradish peroxidase complex as the final layer. Peroxidase was demonstrated using 3,3'-diaminobenzidine. The biotinylated tyramine signal amplification method resulted in the sensitive detection of immunonoglobulin light chain mRNA, with the whole procedure being completed in one day. Moreover, the use of peroxidase as the final reporter molecule also allowed haemamatoxylin to be used as counterstain, thereby permitting the evaluation of cellular morphology.     

Detection of immunoglobulin light chain mRNA by in situ hybridisation using biotinylated tyramine signal amplification.

A highly sensitive method for the light microscopic in situ hybridisation of immunoglobulin light chain mRNA in formalin fixed, paraffin wax embedded sections is reported. This method is based on signal amplification using horseradish peroxidase catalysed deposition of biotinylated tyramine at the sites of hybridisation. kappa and lambda light chain immunoglobulin mRNA in situ hybridisation was performed with fluorescein isothiocyanate conjugated oligonucleotide probe cocktails. The hybridisation signal was detected using a biotinylated tyramine signal amplification procedure with streptavidinbiotin-horseradish peroxidase complex as the final layer. Peroxidase was demonstrated using 3,3'-diaminobenzidine. The biotinylated tyramine signal amplification method resulted in the sensitive detection of immunonoglobulin light chain mRNA, with the whole procedure being completed in one day. Moreover, the use of peroxidase as the final reporter molecule also allowed haemamatoxylin to be used as counterstain, thereby permitting the evaluation of cellular morphology.     

Apoptosis of colon cancers assessed by in situ DNA nick end-labeling method

Apoptosis in colon cancers was investigated using in situ DNA nick end-labeling. Seventy-six colon cancer cases were divided according to histologically defined malignant grading, degree of invasion and other pathologic indicators. The apoptotic labeling index (ALI) was highest in cases with invasion limited to the submucosa, the difference as compared to other stages being statistically significant, while no correlation was noted between ALI and histological type. ALI significantly decreased with lymph node involvement of cancers. In contrast, there appeared to be no link with vessel invasion. ALI was significantly higher in lesions smaller than 2 cm in diameter. These results indicate that apoptosis in colon cancers is more frequent at a relatively early stage when the lesions are small, and suggest that the apoptotic phenomenon may play some role in regulating the size and progression of such tumors.     

Detection of human papillomavirus in matched cervical smears and biopsy specimens by non-isotopic in situ hybridisation.

AIMS: To determine the relative diagnostic sensitivity of non-isotopic in situ hybridisation (NISH) for the diagnosis of human papillomavirus (HPV) on matched smears and biopsy specimens; to compare the NISH signal type in the two samples; and to correlate the NISH data with the morphological diagnosis. METHODS: HPV samples were assayed individually by NISH with digoxigenin labelled probes (HPV6, 11, 16, 18, and 33) on routinely collected paraffin wax embedded cervical biopsy specimens and for high risk HPVs with a cocktail of similarly labelled probes (HPV16, 18, 33) on matched smears. These were taken at the same colposcopic examination from 32 patients investigated for an abnormal cervical Papanicolaou (PAP) stained smear. RESULTS: An HPV signal was present in 18 (56%) biopsy specimens and in 14 (44%) smears. There was higher concordance of sets of data in the presence of cytopathic wart virus changes. The superiority of biopsy over smear in detecting HPV was mainly the result of examining the entire cervical biopsy specimen rather than cells scraped from the cervical surface. The NISH signal type in both biopsy specimen and smear was similar; it has been shown that NISH type 1 signal correlates with episomal viral replication and type 2 and 3 signals with viral integration. CONCLUSIONS: These data show that NISH on cervical smears is a worthwhile primary screen for HPV infection. The NISH signal types in cervical smears are similar to those previously described in cervical biopsy specimens.     

Detection of parvovirus B19 in macerated fetal tissue using in situ hybridisation.

AIMS: To compare the application of a non-radioactive in situ hybridisation (ISH) technique with an immunocytochemical technique for the detection of human parvovirus B19 in formalin fixed, paraffin wax embedded sections of macerated fetal tissue. METHODS: Archived samples of liver, lung or kidney from 19 human fetuses were investigated for parvovirus B19 using a full length digoxigenin labelled DNA probe of 5.5 kb; bound probe was detected using an anti-digoxigenin (alkaline phosphatase) conjugate and visualised using NBT/BCIP. Immunocytochemical detection of parvovirus B19 was performed using a monoclonal mouse antiparvovirus B19 antiserum, with a streptavidin-biotin complex (horse radish peroxidase) method. Cases were selected to provide a range of diagnostic certainty and a range of degrees of macerative degeneration. RESULTS: Parvovirus B19 was found in 15 of 19 cases using the B19 ISH technique compared with 8 of 19 cases using the immunocytochemical technique. The four negative cases were all controls known to be parvovirus B19 free. All ISH positive cases showed excellent staining with low background regardless of extent of maceration and tissue type. In comparison, sections stained by the immunocytochemical method showed considerable non-specific immunoreactivity in many cases, particularly with severe maceration. Kidney and lung tissues gave the cleanest results. CONCLUSIONS: ISH is more effective than the immunocytochemical technique for the detection of human parvovirus B19 in macerated fetal tissue. The lack of detectable background staining with the ISH technique led to easier interpretation suggesting that this technique should be the method of choice for the investigation of parvovirus B19 in macerated postmortem tissues.     

Familial complex chromosome rearrangement ascertained by in situ hybridisation.

A complex familial chromosome translocation has been ascertained by combining classical cytogenetics and CISS (chromosomal in situ suppression). Cytogenetic analysis of a chorionic villus sample with G banding showed a 47,XX,-2, +der(2)t(2;22),+der(22)t(2;22) karyotype. Analysis of peripheral blood lymphocytes from the parents by G banding and CISS showed a more complex translocation in the father: 46,XY,-2,-11,-22, +der(2) t(2;11)(q13;q23), +der(11) t(11;22) (q23;q11.2), +der(22) t(2;22) (q13;q11.2). Definitive analysis of cultured amniotic fluid cells showed a double partial trisomy of chromosomes 11 and 22. The couple decided to continue the pregnancy. The fetal karyotype was confirmed at birth. Clinical abnormalities present in our patient were typical of an unbalanced 11;22 translocation. Our findings confirm that chromosome painting techniques allow a better characterisation of complex chromosome rearrangements which may be difficult to detect in G banded karyotypes.     

Comparative analysis of human papillomavirus detection by dot blot hybridisation and non-isotopic in situ hybridisation.

AIMS: To determine the relative diagnostic performance of non-isotopic in situ hybridisation (NISH) and a dot-blot assay for detecting human papillomavirus (HPV) on exfoliated cervical cells; and to correlate the results with cytopathological assessment. METHODS: Cervical smears and cytological samples were obtained from 122 patients during the same clinical examination and the presence of HPV sequences determined by NISH and dot-blot analysis, respectively. RESULTS: Dot-blot analysis gave an autoradiographic signal in 15 of 121 (12.4%) cases, while NISH detected viral genomes in 38 of 114 (33.3%) cases. Even in the presence of koilocytosis, where vegetative replication of the virus occurs, NISH was positive in over twice as many cases as dot-blot analysis (NISH 90%, dot-blot 40%), while in smears within normal cytological limits, where the viral copy number is likely to be considerably lower, the differences were more striking (NISH 31%, dot-blot 5%). CONCLUSIONS: These data show that NISH on cytological smears is more sensitive than a standardised dot-blot hybridisation assay for detecting HPV infection in cytological material and is therefore a more appropriate screening tool.     

Detection of enterovirus RNA in experimentally infected mice by molecular hybridisation: specificity of subgenomic probes in quantitative slot blot and in situ hybridisation

Subgenomic cDNA clones representing defined regions of the genome of Coxsackie B3 virus were used as hybridisation probes to detect RNA of various enteroviruses in cell culture and mouse model systems. Radiolabelled probes were used in slot blots to quantitate the RNA in samples; biotinylated probes were used to localise virus RNA at the cellular level by in situ hybridisation with monolayers of infected cells or thin sections of tissue samples. Probes derived from the 5' or 3' terminal regions of Coxsackie virus RNA, which are highly conserved in the genus Enterovirus, detected RNA of various serotypes in infected cell cultures, but failed to hybridise with hepatitis A virus (HAV) RNA. Although HAV is clearly a Picornavirus, our data support the view that its taxonomic position within the enteroviruses should be reconsidered. The biotinylated probes were also used to detect in situ virus RNA in paraffin-embedded tissue samples from experimental mouse models of Coxsackie B3 virus-induced myocarditis or Coxsackie B1 virus-induced myositis. Since the integrity of the tissues was preserved during the process, and viral RNA was localised in the affected muscle fibres, this has enabled us unequivocally to relate the infecting virus to the underlying tissue injury.