HIN-1 expression in human tissues. (A, B, C) Representative mRNA in situ hybridization using digitonin-labeled human HIN-1 antisense ribo-probe on normal (A, ×20, B, ×200 magnification) and DCIS (C, ×200 magnification) mammary epithelium. Hybridization with the sense probe gave no signal (data not shown). (D) Evaluation of HIN-1 expression in 76 human tissues on a dot blot expression array. High level of expression was detected in breast (BR), lung (LU), esophagus (ES), duodenum (DU), trachea (TR), prostate (PR), salivary gland (SG), fetal lung (FL), and fetal kidney (FK), whereas lower level expression was seen in pancreas (PA), lymph nodes (LN), nucleus accumbens (NA), and pituitary gland (PI). Arrow indicates a column of cancer cell lines including leukemias, lymphomas, lung, colorectal, and cervical cancer cell lines. (E) Analysis of HIN-1 expression on multiple tissue Northern blots to confirm the size of the hybridizing RNA.

Putative HIN-1 homologues. (A) Amino acid alignment of human, mouse, and rat HIN-1 proteins. Identical and conserved amino acids are highlighted; shading intensity correlates with homology. N-terminal signal peptide and predicted signal peptidase cleavage site are indicated by underlining and arrow, respectively. (B) Phylogenetic comparison of HIN-1 homologues. Comparisons were made by using dnastar and the Jotun Hein algorithm. Most of the HIN-1-related proteins do not have full-length sequence; therefore the degree of similarity may be imprecise.

HIN-1 expression in breast carcinomas and breast cancer cell lines. (A) Northern blot analysis of HIN-1 expression in normal human mammary organoids (freshly isolated breast ducts), myoepithelial cells (HME), mammary epithelium from a 25-wk-pregnant patient (Preg), and various breast cancer cell lines. The weak actin hybridization signal in the pregnant mammary tissue sample (Preg) is likely due to partial RNA degradation. (B) RT-PCR analysis of HIN-1 expression in LCM (laser capture microdissected) purified breast cancers and corresponding normal epithelium. Numbers indicate case numbers, whereas “is” and “T” denote in situ and invasive carcinomas, and “ad” atypical ductal hyperplasia. Amplification of the β-actin cDNA was used as control. (C) Real-time PCR analysis of HIN-1 expression in LCM-purified primary breast carcinomas and corresponding normal epithelium. Fold change indicates the ratio of HIN-1 mRNA levels in normal and cancerous epithelium. Numbers indicate case numbers, whereas “is” and “inv” denote in situ and invasive carcinomas, and “adh” atypical ductal hyperplasia.

Analysis of methylation patterns in the HIN-1 promoter region. (A) HIN-1 proximal promoter region and first exon. Transcription and translational initiation sites are indicated with arrow and ATG, respectively. Bars represent potential methylation sites (CpG). Arrows indicate the location of forward (F) and reverse (R) primers used in methylation-specific PCR reactions. (B) Results of sequence analysis of bisulfite-treated genomic DNA from the indicated cell types. ZR75-1-AC indicates 5azaC-treated cells. Circles represent potential methylation sites (CpG), whereas shading intensity indicates the frequency at which the site was found to be methylated in the clones analyzed (10–100%). HIN-1 mRNA levels are indicated by “+” and “−” signs; +++ denotes high level of expression detected only in normal luminal epithelial cells; + indicates mRNA levels detectable by Northern blot analysis of 5 μg of total RNA. (C) RT-PCR analysis of HIN-1 expression levels before and after 5-aza-deoxy-cytosine (5azaC) treatment in the indicated cell lines and in untreated/uncultured normal mammary epithelium (N). Amplification of the β-actin cDNA was used as control. (D) Methylation-specific PCR analysis of the HIN-1 promoter region in primary tumors (Upper) and breast cancer cell lines (Lower). M and U indicate amplification using methylated and unmethylated sequence-specific primers, respectively. DNA prepared from ZR-75-1 (ZR75-1) breast cancer cell line and normal (N) mammary tissue were used as controls. Most of these primary tumors were not microdissected; therefore amplification with the unmethylated primers could be due to contaminating normal tissue or could be due to tumor heterogeneity. (E) Luciferase activity in HIN-1-methylated and unmethylated cells after transient transfection with promoterless pBR-pl-luc or pBR-pl-HIN-1prom-luc plasmid.

HIN-1 is a secreted protein that negatively regulates cell growth. (A) Western blot analysis of HIN-1 protein expression. Cells and media from 293 cells (first four lanes) transfected with pCEP4 (C), pCEP4-His-HIN-1 (H) constructs, and MCF10A or SUM159 cells infected with Ad-Track-GFP (G) or Ad-Track-His-HIN-1 (H) were lysed in denaturing urea buffer and incubated with Ni-nitrilotriacetic acid (NTA) beads. Bound proteins were resolved by PAGE and immunoblotted with polyclonal rabbit anti-human-HIN-1 antibody. (B) Results of a representative colony growth assay experiment in BT549 and MDA-MB435 cells. Cells transfected with empty vector (pCEP4), HIN1, or p53 expression constructs were selected for 2 weeks, followed by crystal violet staining. Hundreds of colonies were observed in control (pCEP4) flasks in both cell lines; no colonies were detected in p53 transfectants. HIN-1 expression significantly suppressed colony growth in BT549 cells and to a lesser degree in MBA-MD435 cells.