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The early and frequent occurrence of deletions at 3p21.3 in lung cancer has led to the consideration of this chromosomal region as a lung cancer (LUCA) critical region with tumor suppressor activity. We covered this 19 genes-containing region with overlapping P1 artificial chromosomes (PACs), in which genes are likely accompanied by their own promoters or other regulatory sequences. With these PACs we transfected cells from a small cell lung cancer (SCLC) cell line which readily caused tumors in nude mice. Per PAC we selected two cell clones with a low number of PAC copies integrated at a single genomic site. The selected clones were s.c. injected into nude mice to investigate whether the integrated genes suppressed the tumor-inducing capacity of the original SCLC cell line. We could demonstrate PAC-specific gene expression in the transfected cells. All of the PAC integration sites were different. It appeared that introduction of a PAC or even an empty PAC vector causes some chromosomal instability, which in principle may either promote or inhibit cell growth. However, both cell clones with integration of the same PAC from the centromeric part of the LUCA region in different genomic sites were the sole pair of clones that caused smaller tumors than did the original SCLC cell line. This suggests that rather than the induced chromosomal instability, the DNA sequence of that PAC, which in addition to two protein-encoding genes contains at least one potential miRNA gene, is responsible for the tumor suppressor activity., ((c) 2006 Wiley-Liss, Inc.)