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Aims The study intended to reveal whether HPV infection is reflected by nuclear morphology and DNA cytometry parameters in head and neck squamous cell carcinomas (HNSCC). Methods In total, 39 HNSCC were selected for reanalysis by histomorphology applying the core classification, DNA cytometry and HPV detection. For the core classification, HE sections were assessed by a score system to evaluate the nuclear size, the mitosis size, their variabilities and the presence of tripolar or tetrapolar mitoses. HPV was analyzed by consensus PCR followed by a hybridization method for virus typing. Static DNA cytometry was applied on single cell suspension focusing particularly on the parameters DNA modal value, DNA index peak, DNA index mean, 2c deviation index and 5c exceeding rate. Statistical analysis was done by T -test or Fisher's exact test. Results The analysis revealed that HPV positive HNSCC had significantly smaller nuclei than HPV negative cases. Increasing values of the nuclear size and mitosis size were significantly associated with higher indices of the DNA cytometry analysis. Conclusions The study confirms that the core classification can provide information on the ploidy of HNSCC and that HPV positive tumors represent a distinct morphological and genetic carcinoma subtype.

The PITX1 (pituitary homeobox 1) gene has essential roles in human development and has been considered a tumor suppressor in various cancers. However, in lung cancer the role of PITX1 remains to be elucidated. In this study, we analyzed the expression of PITX1 at both mRNA and protein levels in human lung cancer. The reduced PITX1 expression was found in cancer cell lines test compared to normal human bronchial epithelia cells (HEBC) and small airway epithelia cells (SAEC) by Northern blot analysis and RT-PCR as well as Western blot analysis. In primary lung tissues, PITX1 mRNA was found to be downregulated in the majority of tumors compared with normal lung tissues. An association between the lack of PITX1 mRNA expression and higher tumor grade was observed. A tissue microarray containing 135 primary lung carcinomas was analyzed by immunohistochemistry. Eighty-four cases (62%) exhibited no expression of PITX1 and the lower expression of PITX1 was significantly linked to higher tumor stages. Additionally, PITX1 was found to be upregulated in lung cancer cell lines H2228 and H526 after they were exposed to a differentiation modifying agent 5-bromodeoxyuridine (BrdU). Since homeobox genes are known to transcriptionally regulate key cellular processes and associated with differentiation and carcinogenesis, we suggest that PITX1 might be linked to lung cancer development and progression.

Frequent allelic loss of the chromosomal region 17p13 in breast cancer has suggested that more tumor suppressor genes, besides p53, are located in this region. By doing suppression subtractive hybridization to detect differentially expressed genes between the breast cancer cell line CAL51 and a nontumorigenic microcell hybrid CAL/17-1, we identified the gene for the γ-aminobutyric acid type A (GABAA) receptor associated protein (GABARAP), located on 17p13.1. GABARAP displayed high expression levels in the microcell hybrid CAL/17-1 but only weak expression in CAL51 and other breast cancer cell lines tested. Furthermore, we observed large vesicles in CAL/17-1 by immunofluorescence staining, whereas no signal could be detected in the tumor cell line. GABARAP mRNA expression and protein expression were significantly down-regulated in invasive ductal and invasive lobular carcinomas compared with normal breast tissue measured by semiquantitative reverse transcription–PCR and immunohistochemistry, respectively. We assessed that neither mutations in the coding region of the gene nor hypermethylation of CpG islands in the promoter region are responsible for loss of gene expression in CAL51; however, 5-aza-2′-deoxycytidine treatment was effective in gene up-regulation, suggesting a methylation-dependent upstream effect. Stable transfection of GABARAP into CAL51 resulted in an increase of gene expression and remarkably influenced the ability of colony formation in soft agar and the growth rate in vitro and, moreover, suppressed the tumorigenicity of the cells in nude mice. In summary, our data suggest that GABARAP acts via a vesicle transport mechanism as a tumor suppressor in breast cancer.

Cell-Cell communication via gap junctions plays a key role in carcinogenesis and in growth control. One of the gap junction proteins, Connexin 26 (Cx26) was considered as tumor suppressor in various cancers. In our study, the expression of Cx26 was analyzed in human lung cancer. The reduced mRNA expression was observed in 17 lung cancer cell lines examined by Northern blot analysis and RT-PCR. In 138 primary carcinomas comprising all subtypes analyzed by immunohistochemistry, 85 cases (62%) exhibited no expression of Cx26, whereas in other 53 cases the Cx26 staining was positive (38%). Additionally, an association between Cx26 protein expression and higher grading of tumors was found in whole tumor samples (p =0.028) but no statistically significant correlations could be observed with tumor stage, tumor size and node status. In squamous cell carcinoma, tumors with higher stage and grading were linked to higher expression of Cx26 (p = 0.015 and 0.017, respectively). To explore the mechanism responsible for the downregulation of Cx26, we treated 2 lung cancer cell lines H2170 and H226 with the demethylation agent 5-aza-2'-deoxycytidine and found the reexpression of Cx26 mRNA. Methylation status of these 2 cell lines was further analyzed by PCR amplification of bisulfite modified DNA and sequencing. A heterogeneous methylation pattern turned out. Our results suggest the inactivation of Cx26 in lung cancer may be explained by promoter methylation.

Suppression subtractive hybridisation (SSH) was performed comparing normal bronchial epithelial cells with a lung squamous cell carcinoma (SCC) and a metastatic small-cell lung carcinoma (SCLC). The sequence analysis of four cDNA libraries revealed 869 individual sequences. Of these, 342 were tested using northern blots of lung cancer cell lines representing the three major subtypes (SCC, adenocarcinoma, SCLC) which confirmed the differential expression of 236 cDNAs. The extended analysis of 31 randomly chosen fragments confirmed the validity of the approach to identify genes associated with lung cancer development. Additionally, five novel full-length cDNA were isolated encoding the microtubule-associated proteins 1A/1B light chain 3, the epithelial V-like antigen 1 (EVA1), the GTP-binding protein SAR1, a new member of the S100-type calcium binding protein family and a new homeobox-containing gene.

Suppression subtractive hybridization (SSH) was applied to identify differentially expressed genes in the SV40LT immortalized human bronchial epithelial cell line Y-BE, with normal human bronchial epithelial cells (HBEC) as a control. Two cDNA libraries of up- and downregulated genes were generated, comprising 218 known genes and 131 unknown genes in total. The expression of 22 clones from the 2 libraries was investigated by Northern blot analysis, and 86.4% (19/22) of them showed differential expression between Y-BE cells and HBEC. Although the Y-BE cells are nontumorigenic in nude mice, Comparative genomic hybridization (CGH) detected some DNA imbalances in Y-BE cells that were similar to lung cancer cells. Our data demonstrate that the studied cell line Y-BE and SSH is a reliable approach for identifying new genes that are associated with immortalization and early tumor development that may help to understand the pathogenesis of lung cancer.

Objectives: The expression patterns of cytokeratin (CK) filaments in human epithelial neoplasms are complex and distinctive. The aims of this study were to analyze CK expression and to evaluate the diagnostic application of CKs in human non-small cell lung cancer (NSCLC). Methods: mRNA expression of CK5, CK6, CK14, CK15, CK17, and CK19 was analyzed by Northern blotting. Protein expression of CK5/6, CK7, CK14, CK17, and CK18 was evaluated by immunohistochemistry on tissue microarrays. Results: Northern blotting showed that CKs were highly expressed in human bronchial epithelial cells and/or small airway epithelial cells. In NSCLC cell lines, the expression pattern of CKs was heterogeneous. Regarding protein expression of CKs in 95 primary lung tumors, expression of CK5/6, CK14, and CK17 proteins was increased in squamous cell carcinomas compared to adenocarcinomas (ADC; p = 0.001, p = 0.030, and p = 0.001, respectively), and higher expression was significantly associated with lower grading (p = 0.006, p = 0.002, and p = 0.001, respectively), while increased expression of CK7 and CK18 was observed in ADC (p = 0.001, respectively). Conclusions: Our data suggest that CK5/6, CK7, CK14, CK17, and CK18 have diagnostic value in the subclassification of NSCLC.

The homeobox containing gene HOP (Homeodomain Only Protein) was identified in the developing heart and lung where it functions downstream of Nkx2.5 and Nkx2.1 to modulate cardiac and lung gene expression. Previously, we found that HOP was downregulated in lung cancer. In this study, we constructed an expression vector containing the full-length cDNA of HOP and transfected it into a lung cancer cell line H2170. Stable transfection led to an increased expression of HOP confirmed by Northern blot analysis. HOP positive transfectants remarkably reduced the growth rate and the ability of anchorage-independent growth in soft agar, and moreover suppressed the tumor formation in nude mice compared to controls. Transient transfection of Nkx2.1 into H2170 resulted in the overexpression of HOP, and correspondingly, siRNA silencing of Nkx2.1 reduced the expression of HOP in lung cancer cells. Treatment with a differentiation modulating agent 5-bromodeoxyuridine (BrdU) led to restoration of HOP expression in a small cell lung cancer cell line H526. In 29 paired primary lung tumor samples, loss of heterozygosity (LOH) analysis was performed by using the 3 microsatellite markers D4S189, D4S231 and D4S392 around the region of chromosome 4q12 where HOP locates. LOH was only found in 4 out 23 cases (17.4%) indicating that allelic loss is a rare genetic event not responsible for the downregulation of HOP in lung cancer. Taken together, our data suggest that HOP is a potential tumor suppressor possibly involved in lung cancer differentiation, and functions downstream of Nkx2.1.

Insulin-like growth factor binding protein 7 (IGFBP-7) is considered a tumor suppressor in various cancers, but its role in colorectal cancer (CRC) is still uncertain. The aims of this study were to analyze the IGFBP-7 expression, and explore the mechanism responsible for the inactivation of IGFBP-7 in CRC.mRNA expression was studied by RT-PCR and Northern blot analysis of cultured cells. Methylation status was analyzed by treatment with 5-aza-2'-deoxycytidine followed by sequencing of PCR products of sodium bisulfite-treated genomic DNA. IGFBP-7 protein expression was evaluated by immunohistochemistry (IHC) on tissue microarrays.mRNA expression was lost in six out of eight CRC cell lines as compared to normal colon cells. DNA methylation was found in the region of exon 1 and intron 1 of IGFBP-7. In tumor tissue, 107 out of 279 samples showed a negative expression of IGFBP-7 by IHC, which was significantly associated with poor prognosis. The analysis of 37 paired cancerous and normal mucosa samples confirmed the downregulation in the tumors, but revealed variable basal expression levels of IGFBP-7 in normal mucosal samples.DNA methylation is a mechanism responsible for IGFBP-7 gene silencing providing a target for therapeutic intervention of this tumor suppressor gene.

In this study we investigated the RNA helicase A (RHA) gene localized on chromosome 1q25 in 15 lung cancer cell lines, 54 primary carcinomas, bronchial epithelial cells and normal lung. Both RT-PCR and real-time RT-PCR showed that RHA is over-expressed in tumour samples compared to normal lung tissues (p=0.01). There was a tendency for higher expression levels in small cell lung cancer compared to non-small cell carcinomas. Over-expression of Y-162 correlated with high grade tumours (p=0.023). However, there was no correlation with tumour stage and survival. Our observations are compatible with the role of RHA in lung cancer development.

We have isolated a novel gene, lung cancer-associated gene Y (LAGY), by suppression subtractive hybridization. The nucleotide sequence of LAGY predicts a small protein of 73 amino acids containing a putative homeobox domain with a molecular mass of 8.1 kD. Multiple-tissue Northern blot analysis revealed that LAGY is present in human placenta, lung, brain, heart and skeletal muscle. Gene mapping locates LAGY on chromosome 4q11–13.1. The expression of LAGY mRNA was widely lost in 18 lung tumor cell lines comprising all major histological types, as shown by Northern blot analysis and semiquantitative reverse transcription-polymerase chain reaction. In an investigation of 72 primary lung tumors, this gene was significantly downregulated in tumors compared to 9 normal lung tissue samples. There was a significant reduction of LAGY expression in squamous cell carcinoma (SCC; n = 27) with increasing grade and stage. No expression was detectable in two high-grade SCCs or two small cell and large cell lung carcinomas (n = 4 for each). In adenocarcinoma (n = 37), expression was reduced; however, this did not reach statistical significance. Since homeodomain-containing genes are known to transcriptionally regulate key cellular processes and are associated with carcinogenesis, we suggest that LAGY might be linked to lung cancer development and progression.

Invasive breast carcinomas are characterized by a complex pattern of chromosomal alterations. We applied comparative genomic hybridization (CGH) to analyze 105 primary breast carcinomas using histograms to indicate the incidence of DNA imbalances of tumor subgroups and difference histograms to compare invasive ductal carcinomas (IDC) with lobular carcinomas (ILC), well and poorly differentiated carcinomas (G1/G3) and estrogen receptor-positive and -negative tumors (ER+/ER−). Only single imbalances showed a higher incidence in ILC compared with IDC, i.e., gains on chromosomes 4 and 5q13–q23 as well as deletions on chromosomes 6q, 11q14-qter, 12p12-pter, 16q, 17p, 18q, 19, and 22q. Of these, particularly gains of 4 and losses at 16q21–q23, and 18q12–q21 were statistically significant. For most loci, IDC showed more alterations providing a genetic correlate to the fact that ductal carcinoma overall is associated with a worse prognosis than ILC. Of these, many imbalances showing statistical significance were also observed in G3 and ER− tumors, i.e., deletions at 2q35–q37, 3p12—p14, 4p15–p16, 5q, 7p15, 8p22–p23, 10q, 11p, 14q21–q31, 15q, and gains at 2p, 3q21–qter, 6p, 8q21-qter, 10p, 18p11–q11, and 20q, suggesting that they contribute to a more aggressive tumor phenotype. By contrast, gains on chromosome 5q13–q23 as well as deletions at 6q, 16q and 22q were more prevalent in G1 and ER+ tumors. The ratio profiles of all cases as well as histograms are accessible at our CGH online tumor database at http://amba.charite.de/cgh. Our results highlight distinct chromosomal subregions for cancer-associated genes. In addition, these imbalances may serve as markers for a genetic classification of invasive breast cancer. Int. J. Cancer 89:305–310, 2000. © 2000 Wiley-Liss, Inc.