Introduction

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When differentiation is associated with irreversible exit from the cell cycle it is called terminal differentiation (1). Terminal differentiation and apoptosis are physiologic mechanisms for regulating the number of cells within continually regenerating tissues, such as epithelia. Terminal differentiation into ciliated cells is a normal process in human bronchial epithelium (2). In contrast, squamous metaplastic terminal differentiation occurs in response to injury, including physical injury or exposure to inhaled carcinogens (3). When normal bronchial epithelial cells (BEC) are placed in culture using typical culture conditions (7) they undergo squamous metaplastic differentiation. This process is accelerated when the population reaches confluence (7). In addition, incubation of normal BEC in medium containing serum (8) or tetradecanoylphorbol 13-acetate (TPA) (9) is associated with induction of terminal differentiation. The process of terminal squamous differentiation is best understood in epidermal keratinocytes (10). During terminal squamous differentiation, a layer of proteins is deposited on the inside of the cell membrane to form a crosslinked, cornified cell envelope (1). The small, proline-rich protein (spr1) expression that is associated with squamous differentiation (13) serves an important function in linking together loricrin proteins during formation of the crosslinked envelope (14). Further, expression of spr1 messenger RNA (mRNA) is inducible in epithelial cells by TPA and ultraviolet radiation (15, 16). Because spr1 expression is inducible and is an important component of the cornified cell envelope, we hypothesized that somatic cell mutations associated with altered expression of spr1 may disrupt terminal differentiation in squamous metaplastic BEC, thereby contributing to the malignant transformation process. Thus, mRNA expression of spr1 was analyzed in normal and malignant BEC lines incubated in media that were optimal for proliferation (BEC growth medium [BEGM] for normal BEC populations and RPMI-1640 containing 10% fetal bovine serum [FBS] for malignant BEC lines). In addition, normal cells were exposed to RPMI-1640 containing 10% FBS to compare gene expression in normal and malignant cell populations under identical growth conditions. Furthermore, spr1 mRNA levels were analyzed in normal and malignant BEC populations grown to confluence. It was determined that spr1 is expressed at high levels in each of the five normal BEC cultures and its expression is induced under conditions that induce terminal differentiation, including confluence and exposure to serum-containing medium. In contrast, spr1 mRNA was absent or its levels too low for quantification in all 12 carcinoma cell lines evaluated under subconfluent as well as confluent conditions. It is hypothesized that downregulation of spr1 mRNA expression was selected for malignant transformation of the cell lines evaluated because it was mechanistically associated with disruption of squamous metaplastic terminal differentiation.

	Materials and Methods

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Reagents

The reagents, primers, competitive template (CT) mixtures, and cell populations are described elsewhere (17).

Methods

RNA extraction; reverse transcription; quantitative reverse transcriptase polymerase chain reaction (RT-PCR) measurement of gene expression; methods for statistical analysis of the data; and conditions for culturing normal BEC lot numbers 10525, 17378, 17684, 6F0333, and 6F0450 (Clonetics, San Diego, CA), as well as malignant BEC lines H446, H82, N417, A549, A427, H2126, Calu-1, SW900, H520, H322, H661, and H460 (American Type Culture Collection, Rockville, MD) were all previously described (17).

To analyze confluent cells, RNA was extracted from normal and malignant cultures (incubated in BEGM and RPMI-1640 containing 10% FBS, respectively) 2 to 3 d after reaching confluence. Note that cell lines H82 and N417 grow as suspension cultures, and thus RNA from these samples was not obtained from confluent cells.

PCR Amplification of Genomic DNA

Genomic DNA was purified from cultures as described in the TRI-REAGENT manufacturer protocol. The same primers that were used in amplifying spr1 sequences from complementary DNA (cDNA) were also used for amplification of 0.5 to 5 µg genomic DNA. PCR reaction conditions were the same as previously described for RT-PCR (17).

	Results

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Expression of spr1 mRNA in Normal and Malignant BEC Cultures

The spr1 mRNA expression, along with that of 26 other genes, including two housekeeping genes, was measured in normal and malignant BEC cultures. Results from representative experiments that included cDNA from subconfluent normal BEC lots 6F0333, 10525, and 17378 grown in BEGM, and subconfluent malignant cell lines H82, A549, and SW900 grown in RPMI-1640 containing 10% FBS are shown in Figure 1. In all reactions, cDNA that was balanced as described (17) was included. Briefly, the concentration of balanced cDNA was such that 1 µl could contribute sufficient native -actin template to compete equally with 5 × 10¹⁴ M -actin CT included in a quantitative PCR reaction. Thus, a balance refers to amplification of native and competitive templates resulting in approximately equally intense ethidium bromide-stained bands after electrophoresis through an agarose gel. As previously described (17, 18), an approximate balance is sufficient to derive quantitative measurement of gene expression. For all experiments, cDNA in balance with 5 × 10¹⁴ M -actin was also in balance with 10¹⁵ M glyceraldehyde-3-phosphate dehydrogenase (GAPDH) CT. For the normal BEC populations, cDNA samples in balance with -actin CT at 5 × 10¹⁴ M were also in approximate balance with spr1 CT present at 10¹⁵ M (lots 6F0333 and 17684) or 10¹⁶ M (lots 10525, 17378, and 6F0450) in quantitative PCR reactions (Figure 1). The range of expression in these populations was 10³ to 10⁴ spr1 molecules per 10⁶ -actin molecules (Table 1).

View larger version (56K): [in this window] [in a new window]   Figure 1.   spr1 mRNA is quantifiable in cultured normal but not malignant BEC populations. Quantitative competitive RT-PCR reactions were performed as described (17). CT molarities of A5 × 10e  14/G15/O15 (where A is the molarity of the -actin CT, G of GAPDH CT, and O of all other CTs including spr1 in the CT mixture) were included in PCR reactions containing cDNA from normal BEC lot 6F0333 grown to 70-90% confluence in BEGM, and CT molarities of A5 × 10e  14/G15/O16 were included in PCR reactions containing cDNA from normal subconfluent BEC lots 10525 and 17378 grown in BEGM. For reactions containing cDNA from subconfluent malignant cell lines H82, A549, and SW900, CT molarities of A5 × 10e  14/G15/O17 were included. Lane 1 contains pGEM size standard; the arrow points to the 350-bp marker. Lanes 2-4 contain -actin (native = 532 bp, CT = 416 bp), GAPDH (native = 788 bp, CT = 582 bp), and spr1 (native = 371 bp, CT = 285 bp) PCR products, respectively.

In contrast to normal BEC populations, spr1 mRNA was not quantifiable in 12 populations of subconfluent tumor cell lines cultured in RPMI containing 10% FBS. A cDNA sample was considered nonquantifiable for a particular mRNA when quantitative PCR reactions that included the relevant primers resulted in visual CT amplification products on an agarose gel but did not result in discernible native amplification products when the CT initially present in the reaction was included at the lowest concentration that could be amplified to produce a visible band. In the case of spr1 that CT molarity was 10¹⁷ M (Figure 1).

In an effort to determine whether spr1 might be expressed at very low levels in malignant BEC lines, we performed PCR reactions that included 10 to 100 times more cDNA than was used in quantification experiments. The results were not quantifiable with this amount of cDNA because the -actin native template exceeded the -actin CT at 5 × 10¹⁴. Under these PCR conditions, in cDNA from subconfluent malignant BEC lines, a faint spr1 PCR product was observed for H322 but for none of the other 11 lines. On the basis of rough calculations, we estimate that the faint band observed represented less than one transcript per 100 cells.

Induction of spr1 Gene Expression by Serum as Well as Confluence in Normal but Not Malignant BEC

Incubation of normal BEC populations in medium containing 10% FBS was associated with a 2- to 23-fold induction of spr1 mRNA (Table 1). When these populations were grown in serum-free medium but allowed to grow to confluence, spr1 mRNA was induced 13- to 40-fold (Table 1). When malignant BEC were grown to confluence, no quantifiable spr1 expression was observed (using 10¹⁷ M spr1 CT). However, when 10- to 100-fold excess cDNA was included in PCR reactions, faint spr1 native product was observed in H446, H520, and H2126 cell lines. Because the conditions were nonquantitative, it was not possible to determine whether spr1 was induced in these lines. However, because the expression was so low (approximately 1 transcript per 100 cells), it is not likely to be functionally significant.

The Inability to Quantify spr1 Expression Is Not the Result of a Deletion of Genomic Sequences

To confirm the presence of sequences corresponding to the spr1 coding region in genomic DNA from the tumor cell lines, DNA from nine of the malignant cell lines was PCR-amplified using the same spr1 primers that were used for amplification of cDNA (Figure 2). These primers span the entire coding region of the spr1 gene. In all cases analyzed, the expected 371-base pair (bp) PCR product was observed; thus, it is clear that the apparent lack of spr1 expression in the carcinoma cell lines is not associated with a large deletion of sequences within the spr1 coding region.

View larger version (18K): [in this window] [in a new window]   Figure 2.   DNA corresponding to spr1 coding sequences is present in the malignant cell lines. Genomic DNA samples from nine malignant cell lines were PCR-amplified using spr1 primers (Table 1 from Reference 17). pGEM size marker was loaded in lane 1. Lanes 2-10 were loaded with spr1 PCR products amplified from the DNA of the malignant cell lines H82, H460, H661, H322, Calu-1, SW900, A427, A549, and H2126, respectively.

	Discussion

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There is evidence that bronchogenic carcinoma cell lines arise from squamous metaplastic bronchial epithelium (4). Because malignant BEC lines are resistant to terminal squamous differentiation induction by TPA or serum, it has been hypothesized that escape from squamous terminal differentiation is a key step in malignant transformation (8, 9). However, no specific genetic or biochemical mechanism has been identified to explain the phenomenon of resistance to squamous terminal differentiation. The squamous phenotype is characterized by formation of a cornified cell envelope (10, 19, 20), which is produced by crosslinking multiple different proteins on the inside of the cell membrane. Other proteins that are important for formation of the crosslinked envelope and that have been identified include transglutaminases (21), involucrin (22, 23), and loricrin (24). None of these genes have been reported to be altered in all 12 of the carcinoma cell lines evaluated compared with normal cells. Protein products of spr1 crosslink loricrin subunits, which comprise the bulk of the cornified cell envelope (14). The spr1 gene can be induced by squamous differentiation inducers (15, 16) and inhibited by retinol, which also upregulates "mucous cell function" in BEC (13). Thus it is reasonable to hypothesize that disruption of spr1 gene expression would result in resistance to terminal squamous differentiation in malignant BEC.

The apparent loss of spr1 gene expression observed here is a striking finding when it is considered that no other single gene that is related to malignant transformation (e.g., tumor suppressor, apoptosis, cell cycling, differentiation genes, oncogenes) has been reported to be altered in all 12 of the carcinoma cell lines included in this study. Although quantitative or qualitative alteration of expression of some of these genes occurs in some of the lines, no single gene other than spr1 has been shown to be altered in all of them.

Primers spanning the entire coding region of the spr1 gene amplified PCR products of the predicted length (371 bp) from genomic DNA corresponding to the tumorigenic cell lines (Figure 2). Thus the apparent lack of spr1 expression in these lines is not due to a large genomic deletion of sequences within the spr1 coding region. It is possible that point mutations lead to altered splicing, and that the resulting alternative transcripts would not be amplified by the primers used. However, it is more likely that the alteration in spr1 expression in these cell lines is due to decreased levels or binding efficiency of transcription factors that regulate spr1 gene expression. This should be testable by introducing reporter plasmids with the reporter gene regulated by spr1 regulatory regions into carcinoma cells.

The factors that regulate transcription of the spr1 gene currently are being investigated. It is now known that TPA stimulates expression of spr1 by increasing the rate of transcription (15); therefore, it is possible that the apparent lack of spr1 expression in the carcinoma cell lines results from an alteration in transcriptional pathways activated by TPA. Because c-jun acts synergistically with TPA treatment in activation of spr1 transcription (28), it is possible that a defect in this transcriptional activator is responsible.

Alternatively, it has been shown in cultured rhesus monkey tracheobronchial epithelial cells that a decrease in spr1 mRNA in response to retinol was due not to a decline in the rate of transcription but to a decrease in the stability of spr1 message (29). Although the mechanism behind this phenomenon is unclear, the observed lack of spr1 mRNA in the carcinoma cell lines may be due to a similar decrease in mRNA stability. Regardless of the mechanism by which spr1 expression was altered, the data presented here indicate that selection for alteration in spr1 expression has occurred in 12 of 12 bronchogenic carcinoma cell lines.

TPA treatment of normal cultured BEC induces terminal squamous differentiation, thus inhibiting cell division (30). In contrast, TPA-treated carcinoma cell lines (including A549, Calu-1, and SW900, which were studied here) are resistant to TPA-induced terminal squamous differentiation, form few or no crosslinked envelopes, and continue to proliferate (9). Furthermore, confluent conditions also induce terminal squamous differentiation associated with crosslinked envelope formation in normal BEC (7) but not in carcinoma cell lines. This information, together with observations reported here that spr1 expression is induced by confluent culture conditions in normal cultured BEC and that spr1 mRNA apparently is neither expressed nor inducible in carcinoma cell lines, leads us to hypothesize that the observed downregulation of spr1 mRNA levels in the carcinoma cell lines permitted them to avoid terminal differentiation.