Quantitative Reverse Transcription-Polymerase Chain Reaction Measurement of HASH1 (ASCL1), a Marker for Small Cell Lung Carcinomas with Neuroendocrine Features¹ Free

In vertebrates the achaete-scute homologue 1 gene has a neuronal determination function as well as a function in the development of a subset of peripheral cells outside a neuronal context, i.e., in the development of bronchial epithelial cells with NE³ features in the lung (1). These pulmonary NE cells show expression of neural markers such as calcitonin gene-related peptide, synaptophysin, and l-DOPA carboxylase. Evidence that the achaete-scute homologue 1 is causally involved in pulmonary NE differentiation came from experiments with homozygous mammalian achaete-scute homologue 1 (Mash1) mutant mice in which normal development of pulmonary NE cells was absent. NE markers were nondetectable in pulmonary cells, although pancreas and gut NE cell differentiation was normal (1). Constitutive expression of Mash1 and SV40 in double transgenic mice airway epithelial cells causes development of large NE lung tumors (2).

In normal lung tissue, HASH1 expression is restricted to epithelial bronchial NE cells. HASH1 expression is strongly increased in lung cancers with NE features, especially in SCLC, the most lethal form of lung cancer (1, 2, 3, 4, 5, 6). In addition, NSCLCs and bronchial carcinoid show expression of HASH1 that correlates with NE features. Loss of NE features is correlated with loss of HASH1 expression, which shows that constitutive HASH1 expression is obligatory for maintaining the NE phenotype (7). In contrast, terminal differentiation of neurons shows a down-regulation of HASH1 expression in fetal neuroblasts and terminally differentiated neuroblastoma tumors, showing the presence of a different role of HASH1 in this context (7). Because HASH1 expression has proven to be a good marker for tumor progression of NE tumors, reliable methods are needed to analyze HASH1 expression in clinical samples.

Quantitative RT-PCR permits quantification of mRNA expression levels in malignant specimens for which the amount of tissue is limited. Because the expression of transcription factors such as HASH1 in tissue specimens is low, quantification techniques such as Northern blot analysis (3) or RNase protection assays, which have been used until now, cannot be used. In this study, we describe the development and use of a real-time quantitative RT-PCR with hybridization probes (8), which makes it possible to determine the progression stage of pulmonary NE tumors in a highly sensitive way using limited amounts of tissue.

NCI-H187 (ATCC CRL-5804) and NCI-N417 (ATCC CRL-5809) cell lines were maintained in Iscove’s medium with glutamax I (Life Technologies, Inc.) supplemented with 10% fetal bovine serum and antibiotics. High versus low expression of HASH1 in these cell lines correlates with NE features. The NCI-H187 cell line is a classic SCLC cell line that has NE features, including expression of l-DOPA carboxylase (9). The NCI-N417 cell line has no NE features. HASH1 expression levels of the NCI-N417 cell line were nondetectable by Northern blot experiments (3), but were detectable with RT-PCR (4). This cell line was used as a negative control for HASH1 expression.

Samples of SCLC (n = 4), NSCLC (n = 2), and normal lung tissue (n = 2), consisting of normal bronchus, were collected by the Department of Pathology and Pulmonology and immediately frozen after biopsy. A pathologist performed histological examination of the stained tissue sections. Tissues were stored in liquid nitrogen. For each RNA isolation, 10 serial sections of 20 μm were cut and processed immediately. The first and last sections were H&E stained.

Total RNA was isolated from both cultured cells and tissue sections by RNAzol B (Campro Scientific) as described previously (10).

RT-PCR was performed in the Superscript II (RNase H-negative) One Step RT-PCR System (Life Technologies) as described previously (10). Primers and annealing temperatures are listed in Table 1. Gel purification of PCR products was performed using the QIAquick Gel Extraction Kit protocol, and isolated bands of PBGD and HASH1 were subsequently cloned into pPCR-Script Amp SK(+) (Stratagene) and pGEMT (Promega) vectors, respectively. Vectors were sequenced in an ABI 310 Genetic Analyzer (Perkin-Elmer) with Big Dye terminator reactions in combination with gene-specific primers. GenBank Accession numbers for the sequences used were HASH1 DNA sequence NT_009439 and mRNA sequence NM_004316 and PBGD human DNA sequence M95623 and mRNA sequence NM_000190. Plasmids were purified with the Maxiprep anion-exchange procedure (Qiagen). In vitro transcription of cRNA (Promega) was performed according to the manufacturer’s instructions. The in vitro-transcribed RNA (5 μg) was purified by denaturing gel electrophoresis (5% polyacrylamide-8 m urea), followed by band isolation. The gel fragment containing the RNA was cut out, and the RNA was eluted overnight at 37°C in 200 μl of buffer [1× transcription buffer (Promega), 20 units of RQ1 DNase, 0.4 units/μl RNasin]. After the tubes were centrifuged for 4 min at 14,000 × g, the supernatant containing the RNA was removed, transferred to a clean tube, and ethanol-precipitated to further cleanup and concentrate the RNA. The samples were stored at −80°C. PCR was performed to check for DNA contamination.

A standard curve, consisting of serial dilutions of PBGD cRNA, was constructed by RT-PCR using duplicate dilutions. The reaction mixture consisted of 1 μl of in vitro-transcribed PBGD RNA in concentrations from 1 × 10⁹ to 0 copies (diluted in MilliQ water), 3.25 mm Mn(OAc)₂, 0.3 μm each primer (Table 1), 0.2 μm Hybridization Probes 1 and 2 (Tib Molbiol; Table 2), 10 ng of MS2 carrier RNA (Roche), 7.5 μl of RNA LightCycler RNA Master Hybridization probes mixture (Roche), and MilliQ water up to a final volume of 20 μl. The addition of MS2 carrier RNA (10 ng) prevented false-negative results in the lower copy region. The RT-PCR consisted of reverse transcription for 30 min at 61°C, followed by 30 s of denaturation. This was followed by 50 cycles of PCR with denaturation at 95°C for 5 s, annealing at 58°C for 15 s, elongation at 72°C for 9 s (PCR product dependent), and final cooling down to 40°C. Quantitative RT-PCR was performed on the two different SCLC cell lines, using 500 ng of total RNA. The threshold cycle (Ct) at which initial amplification occurs is linearly inversely related to the logarithm of the copy number present in the sample. By interpolation, the exact copy number can be determined. The expression level of HASH1 was quantified by quantitative RT-PCR in the same way as described for PBGD, using HASH1 primers and probes (Tables 1 and 2). When quantitative RT-PCR was performed on clinical specimens (SCLC, NSCLC, and normal bronchus), 100 ng of total RNA was used as template and analyzed as above. In each case, for PBGD as well as for HASH1, reactions were performed at least in duplicate, and gel analysis showed bands of the correct size. The HASH1 expression levels were normalized using the PBGD expression level according to the following equation (the average PBGD expression level of samples n to m is overlined):

In vitro-transcribed cRNA was produced and subsequently analyzed by RT-PCR as a reference for absolute quantification. The housekeeping gene PBGD was used as an internal control to normalize the HASH1 signal. The PBGD gene produces two isoenzymes by use of two transcription start sites. The first produces a broadly expressed transcript. The second start site, located downstream, produces an erythrocyte-specific transcript (11). The first transcript was used as reference. Complete removal of contaminating plasmid was essential for accurate quantification, and this was achieved by overnight incubation with RQ1 DNase One during cRNA purification. Serial dilutions of the purified in vitro-transcribed cRNA were subsequently analyzed by quantitative RT-PCR (Fig. 1,A) and plotted. As is shown in Fig. 2A, a linear standard curve was obtained with a linear range of 100–10⁹ copies of the housekeeping gene PBGD.

Serial dilutions of the HASH1 purified in vitro-transcribed cRNA were analyzed by RT-PCR (Fig. 1,B), and the standard curve showed a linear range between 100 and 10⁹ copies (Fig. 2 B).

The standard curve was used as an absolute quantification reference for quantification of the internal control PBGD mRNA in total RNA of the cell lines NCI-H187 and NCI-N417. As is shown in Fig. 2 C, comparable amounts of PBGD were found when both cell lines were analyzed. The mRNA expression level in cell line NCI-N417 had an average of 3 × 10⁶ copies/0.5 μg of total RNA, and for NCI-H187 the expression average was 5 × 10⁶ copies/0.5 μg of total RNA. All observed values were within the linear range of the standard curve. The variations between samples were between a factor 2 and 5 and are likely to reflect differences in metabolic activities at the time when the cells were harvested.

To evaluate the mRNA expression of HASH1 in SCLC cell lines, a positive NE cell line was used (NCI-H187) and compared with the non-NE SCLC cell line (NCI-N417); NCI-H187 and NCI-N417 have high and a low HASH1 expression, respectively (1, 9). The average HASH1 mRNA expression level in cell line NCI-N417 was 1.2 × 10² copies/0.5 μg of total RNA, and for NCI-H187 the average HASH1 expression was 4.1 × 10⁷ copies/0.5 μg of total RNA (Fig. 2 D). The observed result for the NCI-H187 cell line was within the linear range of the standard curve; however, the observed result for NCI-N417 was near the detection level and false-negative values were observed. There was a relatively small variation in expression levels between samples of different isolation dates, comparable to the variations seen in PBGD expression levels.

Normalized HASH1 mRNA levels are shown in Fig. 2 E. Normalized average mRNA expression levels were 1.8 × 10⁷ copies/0.5 μg of total RNA for cell line NCI-H187 and 3.3 × 10² copies/0.5 μg of total RNA for cell line NCI-N417. This indicates that the difference in normalized HASH1 mRNA expression between both cell lines is at least a factor of 50,000.

The method described above was used to quantify HASH1 mRNA in clinical samples. For this, RNA isolated from SCLC tissues (n = 4) was used and compared with either NSCLC tissue (n = 2) or normal bronchus (n = 2). High HASH1 expression levels were found in all SCLC tissues (Fig. 3,A), when compared with the levels in NSCLC and normal bronchus (Fig. 3 B). The normalized average mRNA expression level in SCLC clinical samples was 2.0 × 10⁶ copies/100 ng of total RNA. In the NSCLC samples, the average normalized expression was 1000-fold lower, i.e., 1.7 × 10³ copies/100 ng of total RNA. The expression in normal bronchus was comparable to the expression levels in the NSCLC, and as such at least 1000-fold lower than in SCLC. These results show that marked and measurable differences exist between SCLCs and other lung tissues (either NSCLC or normal bronchus).

Basic helix-loop-helix factors play a prominent role in the regulation of developmental pathways in normal tissue differentiation in all tissues. During tumor progression, these basic helix-loop-helix genes are switched on in a pattern reflecting their developmental program, but in a situation outside their normal embryonic context. This aberrant expression initiates or maintains a differentiation cascade, which can lead to the establishment of aggressive phenotypes. Here we describe an accurate and reproducible RT-PCR method for quantifying HASH1 mRNA in clinical samples, using the LightCycler fluorescence resonance energy transfer technology. The method is applicable to small biopsy samples, which enables the classification of lung tumors in an early stage and can contribute to diagnosis. Moreover, it can be used for screening of biopsies before and after cancer therapy. Because molecular profiling of tumors can provide information on the clinical status, the method described here can be used as a generic way to make a molecular profile for other transcription factors, including basic helix-loop-helix genes other than HASH1.

HASH1 mRNA signals were normalized using the housekeeping gene PBGD, which is a pseudogene-free housekeeping gene and has minimal transcriptional variability among tissues (11, 12). For amplification of PBGD as well as HASH1, a linear range that spans a region of 10⁷ orders of magnitude was obtained. This allows determination of copy numbers of low-abundance genes, which are expected to be in a range of 0.3 to 1 × 10⁵ copies when 100 ng of RNA are used as input. The positive NE control cell line NCI-H187 showed 50,000-fold higher normalized mRNA expression of HASH1 than did the non-NE cell line NCI-N417, indicating that the method is applicable over a wide dynamic range.

Until now, the expression levels of HASH1 in classic SCLC NE tumors were assayed by Northern blot analysis, and large amounts of poly(A) RNA (2–5 μg) were required because of the relatively low detection level of the method (3). These high amounts of poly(A) RNA can be obtained only from cultured cell lines or large tumors. The same is true for the RNase protection analysis, which requires similar high amounts of RNA. Real-time quantitative RT-PCR, as described here, allows quantification of transcription factor expression levels, using limited amounts of total RNA (100–500 ng), thus enabling quantification with limited amounts of tissue, such as clinical samples. Normalized average HASH1 mRNA expression levels in SCLC clinical samples were 1000-fold higher than in both NSCLC tissue and normal bronchus.

Recently, a member of the Snail family of transcription factors, Scratch 1, has been shown to be correlated with HASH1 expression in NE tumors (4). The Snail family of transcription factors is involved in cell migration, which might suggest that the increased HASH1 expression contributes to the invasive phenotype of SCLC cells. These data further support the model that SCLC is derived from a lung epithelial precursor commitment to the pathway of NE cell differentiation, as claimed by Borges et al. (1) and that HASH1 contributes to the aggressive phenotype.

We would like to thank Thea Tadema and Angelique Verlaan of the Department of Pathology for technical assistance. We also kindly thank Prof. Paul van Diest (Department of Pathology) for critical reading of the manuscript.