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Stromelysin-3 (ST3) is a matrix metalloproteinase which is expressed in fibroblastic cells of most human invasive carcinomas and represents a potential new prognostic indicator. Expression of recombinant ST3 forms in Escherichia coli from cDNA constructs indicated that high levels of expression were achieved when the ST3 pro-domain was deleted. The putative mature form of ST3 thus produced and recovered from bacterial inclusion bodies was used to prepare monoclonal antibodies (MAbs) against ST3 by immunization of BALB/C mice. Ten hybridomas producing MAbs against ST3 were obtained and analyzed for their ability to detect endogenous ST3 in breast cancer and in conditioned media from human fibroblasts. One of these MAbs (5ST-4A9) was found to be suitable for the routine detection of ST3 on breast cancer tissue sections, thus opening the possibility to evaluate ST3 prognostic value in breast cancer using semi-quantitative immunohistochemistry.

MLN 50 was previously identified in a cDNA library of breast cancer metastasis. In this study, we show that MLN 50, which is expressed at a basal level in normal tissues, is overexpressed in 8% of human breast carcinomas most often together with c-erbB-2. MLN 50 cDNA encodes a putative protein of 261 residues, named Lasp-1 (LIM and SH3 protein) since it contains a LIM motif and a domain of Src homology region 3 (SH3) at the amino- and the C-terminal parts of the protein, respectively. Thus, Lasp-1 defines a new LIM protein subfamily.

Using differential screening of a breast cancer cDNA library, we isolated a cDNA encoding the psoriasin (S100A7) protein, previously identified in psoriatic epidermis. In the present study, we demonstrate that the psoriasin gene is expressed in breast cancer cell lines and in cancer cells of some breast carcinomas but not in any non-cancerous tissues examined, except skin. Another S100 gene, S100C, which we co-localized with the psoriasin gene to human chromosome 1q21-q22, was found to be expressed in most tissues and cell lines evaluated. These findings add support to the concept that the S100 genes clustered in human chromosome 1q21-q22 are individually controlled and that some of them may be involved in the regulation of cell transformation and/or differentiation.

A regional survey on peoples' attitudes towards drugs was conducted in the fall of 1992 on 2500 individuals aged 14-70 years. The great majority of those surveyed considered the taking of heroin (98%), cocaine (95.9%) and amphetamines (94.7%) to be "very risky" for one's health. These percentages were considerably lower in the case of tobacco (41.3%) and alcohol (26.7%). Those surveyed pointed to 'heroin' (35.6%), 'all illegal drugs' (27%), 'legal drugs' (17.2%) and 'cocaine' (14.2%) as the most dangerous drugs for society. The study shows how alcohol and tobacco are perceived by Spaniards as less dangerous than illegal drugs.

We describe a differential screening method for cDNA libraries which used a combination of subtracted and PCR-amplified cDNA probes, and which can be applied to the selection of genes expressed in multiple tissues. This technique was used to identify genes commonly overexpressed in breast and basal cell carcinomas. These represent stromally dependent, invasive tumors with and without metastatic capacity. Thus, this screening sought to identify genes involved in the early stages of tumor progression. We identified a total of 16 genes, including c-erbB-2 and tissue inhibitor of metalloproteinases 3 whose products have been implicated in tumorigenesis or invasion. We also identified a novel sequence (D52) showing little homology with others described in any species, which maps to the human chromosomal band 8q21. In situ RNA hybridizations of breast carcinoma sections indicated that the D52 gene was expressed in cancer cells, whereas other genes identified in the differential screening were expressed in fibroblastic or inflammatory cells within the tumor stroma. Thus, the procedure developed in this study selected genes expressed in a diversity of cell types, indicating its potential usefulness in other systems.

Background: Tissue inhibitor of metalloproteinases-3 (TIMP3) is the third member of the TIMP family of proteins, believed to play a significant role in controlling extracellular matrix remodeling., Materials and Methods: Differential screening of a human breast carcinoma cDNA library using substracted and PCR-amplified cDNA probes identified a 4.6-kb TIMP3 cDNA, which was used for further cDNA library screenings, Northern blot hybridizations, and the synthesis of riboprobes for in situ RNA hybridization analyses., Results: The 4.6-kb full-length TIMP3 cDNA contains 3.7 kb of 3'-untranslated sequence. Additional TIMP3 cDNAs subsequently identified were colinear with the original sequence, but revealed use of four different polyadenylation signals within the 3'-untranslated region, which accounted for the 4.6-, 2.7-, 2.5-, and 2.1-kb TIMP3 transcripts noted in this and in previous studies. In situ RNA hybridizations demonstrated that in breast carcinoma the TIMP3 gene was predominantly expressed by fibroblastic cells within the tumor stroma adjacent to cancer cells. TIMP3 transcripts were also strongly detected in fibroblastic decidual cells of pregnant endometrium., Conclusions: Modulating the length of the 3'-untranslated region may represent a mechanism by which TIMP3 gene expression is controlled in tissues. The strong expression of the TIMP3 gene by fibroblastic cells in breast carcinoma supports the importance of tumor stroma as a source of factors influencing human carcinoma growth and progression.

Background: It has long been proposed that secreted proteinases, including the matrix metalloproteinases, play an important part in tumor progression in mediating extracellular matrix remodeling. More recently, it has been suggested that extracellular proteinases also regulate growth factors and cytokines that may contribute to tumor progression., Methods: RNA in situ hybridization and immunohistochemistry were used to study the expression, in breast and other types of human carcinomas, of the stromelysin-3 (ST3) gene, which encodes a putative new member of the matrix metalloproteinase family., Results: The ST3 gene is overexpressed in most types of human carcinomas, including breast carcinoma where ST3 RNA was detected in 95% (99 of 104) of invasive primary tumors. Both ST3 protein and RNA are detected in fibroblastic cells immediately surrounding the cancer cells, but not in the malignant cells or in stromal cells at a distance from them. The ST3 gene also is expressed in some in situ breast carcinomas, where ST3 expression correlates with the known risk of these tumors to become invasive., Conclusions: ST3 is the paradigm of tumor proteinases that are not expressed in the malignant cells of human carcinomas but in fibroblastic cells of tumor stroma. ST3 represents a potential new prognostic parameter to identify subpopulations of aggressive tumors, particularly to evaluate the likelihood of in situ breast carcinoma progression to invasive cancer. Furthermore, the specific expression of the ST3 gene in fibroblastic cells immediately surrounding cancer cells suggests that ST3 may be involved in tumor progression and that it represents a potential target for cancer treatment.

Background: In human mammary tumors, pS2 expression is directly controlled by estrogens and restricted to a subclass of breast carcinomas. In addition, recent studies have suggested that this gene is expressed in both the invasive and preinvasive forms of breast cancer., Experimental Design: pS2 gene expression was investigated in benign and malignant ovary tumors and whenever possible, pS2 expression was also studied in cells collected from cystadenoma fluids. In several cases, particularly with cells from cystadenoma fluids, the limited amount of material available prevented the used of the traditional RNA detection methods such slot/dot blots or Northern blots. Therefore, a rapid and sensitive polymerase chain reaction assay of pS2 expression has been developed and used in this study., Results: In human ovarian tumors, data obtained show that pS2 transcripts and proteins are present in all mucinous cystadenomas studied and at a lower frequency in endometrioid cystadenomas. Quantitation of the CA 19-9 mucin concentration in ovarian fluids indicate that pS2 expression is always associated with high mucin concentrations, but mucin-positive and pS2-negative samples are also frequently observed., Conclusions: These data suggest that pS2 expression is restricted to subclasses of human ovarian cystadenomas.

The ulceration-associated cell lineage (UACL) develops in the human gastrointestinal mucosa after ulceration; it grows out from the bases of adjacent crypts and ramifies in the lamina propria to form a new gland, finally giving rise to a duct by which the glandular secretion and indeed cells are carried to the surface. Using immunocytochemistry and in situ hybridization with 35S-labelled riboprobes, we have defined the pattern of trefoil peptide gene expression (pS2; human spasmolytic polypeptide, hSP), epidermal growth factor/urogastrone (EGF/URO), and the distribution of cell proliferation during the development of the UACL, as indicated by immunostaining for proliferating cell nuclear antigen (PCNA). Our studies reveal that the morphogenesis of the UACL shows a marked morphological resemblance to developing Brunner's glands; the pattern of trefoil peptide gene expression during UACL development is also very similar. However, trefoil peptide gene expression in the mature UACL complex is unique amongst gastrointestinal cells. The mature UACL shows a distinctive proliferative organization: while the early buds and glands are non-proliferative, apparently being fed by cells from the parent crypts, a definitive proliferative zone develops within the duct. This, of course, corresponds to the location of the gastric gland proliferative zone. We propose that while the UACL shows novel features, it shares its differentiation programme with Brunner's glands, but its pattern of cell renewal eventually is that of the gastric gland.

We reviewed 319 primary breast tumours for cytosolic pS2 content, with a median follow-up of 6 years. pS2 status correlated positively with oestradiol and progesterone receptors and negatively with Scarff, Bloom and Richardson grade. pS2 positivity was associated with longer overall survival, particularly in patients who received hormone therapy, in whom pS2 status was also predictive of the response to therapy.

Stromelysin-3 (ST3) belongs to the family of matrix metalloproteinases, a group of proteolytic enzymes which are believed to play a role in tumor invasion and metastasis. In the present study, we report that the ST3 gene, which was initially identified in invasive breast carcinoma, is expressed in most other invasive human carcinomas, but rarely in sarcomas and other nonepithelial tumors. In carcinomas, both ST3 RNA and protein were specifically detected in fibroblastic cells immediately surrounding the cancer cells. In agreement with this observation, the carcinomas which are known to progress without inducing a prominent tumor stroma are also those which usually do not express the ST3 gene. ST3 gene expression was also observed in noninvasive carcinomas of the breast, uterus cervix and bladder, where the probability of detecting ST3 RNA and protein positively correlated with the known risk of these lesions evolving towards invasion. Taken together, these observations further support the hypothesis that ST3 may contribute to tissue-remodeling processes associated with carcinoma progression, and may represent a new prognostic factor to define populations of aggressive tumors.

A new complementary DNA, p27, has been cloned and sequenced from estradiol-treated MCF7 human breast carcinoma cells. It encodes a putative highly hydrophobic protein of 122 amino acids which has a 33% overall sequence similarity to the product of the 6-16 gene (R. L. Friedman, S. P. Manly, M. McMahon, I. M. Kerr, and G. R. Stark, Cell, 38: 745-755, 1984), which is transcriptionally induced by interferons of the alpha/beta type. We demonstrate here that the p27 gene, which is located in band q32 of human chromosome 14, is also induced by interferon-alpha in human cell lines of different origin and that expression is independent of the presence of estradiol receptor in the cells. High levels of p27 RNA were found in vivo in approximately 50% of primary human breast carcinomas (21 were tested by Northern blotting). In situ hybridization to some of the p27-overexpressing tumors showed that the p27 RNA is localized in cancer cells and sometimes also in fibroblastic cells of tumor stroma. p27 RNA levels in the tumors did not correlate with the presence of estrogen receptor or with the expression of the estrogen-induced pS2 gene. Further studies are now necessary to elucidate the cause of p27 gene overexpression in breast carcinoma and in particular to determine whether it corresponds to chromosomal rearrangements in the 14q32 region and/or to induction by interferons of the alpha/beta type.

This study was aimed at assessing the significance of pS2 immunostaining in colorectal adenocarcinoma and adjacent tissue. Paraffin sections of 63 surgically resected colorectal adenocarcinomas were stained with a pS2 specific monoclonal antibody using the avidin-biotin complex immunoperoxidase technique. Several tumor sections also included adjacent nonneoplastic mucosa. Six tubular adenomas and five hyperplasic polyps found in the resected bowels were also examined. Focal staining for pS2 was seen in 32 tumors (51%). In all but one case, less than 10% of the tumor cells were stained. pS2 staining was more common in right-sided than in left-sided tumors (p < 0.05), and was more prevalent in Dukes C than combined Dukes A and B tumors (p < 0.05). No significant relationship was found between pS2 positivity and the degree of tumor differentiation, patients' sex, or outcome of the disease as judged by the development of recurrence or metastasis. Strong pS2 positivity was seen in hyperplastic polyps and in nonneoplastic mucosa adjacent to many tumors. Tubular adenomas were either negative or showed focal superficial staining. It is concluded that pS2 immunostaining in colorectal adenocarcinoma does not seem to have prognostic significance, but may reflect developmental differences between the right and left side of the colon. The presence of pS2 staining in adjacent nonneoplastic mucosa and in hyperplastic polyps suggests that the epithelium in these areas is of a regenerative or reactive nature.

We have previously shown that the human pS2 gene, which codes for a secreted peptide of 60 amino acids, is expressed in a number of human carcinomas, including carcinomas of the breast, the pancreas, and the large bowel. Strong pS2 gene expression was also observed in the normal gastric mucosa and in the regenerative tissues surrounding ulcerous lesions of the gastrointestinal tract. A number of pS2 similar peptides, designated as P-domain peptides, have been described, notably the porcine (PSP), murine (mSP), and human (hSP) spasmolytic polypeptides, which correspond to duplicated pS2 proteins. We have now cloned a mouse homolog of the human pS2 cDNA to dispose of an animal model to study the pS2 protein function, which remains unknown at the present time. We show that the mouse putative pS2 protein sequence and the physiological pattern of expression of the mouse pS2 gene are well conserved. The mouse pS2 gene is highly expressed in the stomach mucosa cells, whereas no pS2 gene expression could be detected in the mouse mammary gland, even during postnatal development processes dependent on growth factors or hormones. Using in situ hybridization, we show that although coexpressed in the fundus, the antrum and the antrum-pyloric regions of the stomach, the mouse pS2 and mSP genes exhibit distinct and complementary cellular patterns of expression.

Alcohol-related mortality and years of potential life lost in Spain in 1986 have been studied according to the official statistics with regard to the population mortality in our country. 6.1% of the deaths in Spain in 1986 were related to alcohol consumption, mainly caused by malignant neoplasm (26.0%), digestive diseases (23.6%), and unintentional injuries (21.1%). Mean potential years of life lost for alcohol-related deaths until 65 was 7.3. Unintentional injuries were responsible for the greater part (61.2%) of alcohol-related years of potential life lost. The present study shows the high mortality rate associated with alcohol consumption in our country, as well as its importance in premature death.

The expression of pS2 was examined histochemically in paraffin sections taken from biopsy material from patients diagnosed with ductal carcinoma in situ (DCIS). Often intense immunoreactivity, to an anti-pS2 monoclonal antibody, was observed in comedo, solid, cribriform and micropapillary types of DCIS, with significant positivity found in 63-67% of cases. In 15 samples analysed, we found a good correlation between pS2 expression and presence of progesterone receptor positive cells, but not with estrogen receptor. There was only a limited degree of correspondence between the cells staining with these anti-sera. Some pS2 positive cells were also seen in normal acini in areas adjacent to cancer but much less frequently in sections of normal breast from reduction mammoplasty. Most normal areas were negative, as were cysts. In benign proliferative conditions (seen in sections with and without DCIS) such as adenosis, sclerosing adenosis, mild and florid ductal epithelial hyperplasia, significant pS2 positivity was seen in about 50% of cases. These results suggest that there is a progressive increase in pS2 from normal to benign to cancer cells and that this gene is expressed in both the invasive and pre-invasive forms of breast cancer.

Matrix metalloproteinases are believed to play an important role in tumor invasion and metastasis. To examine the expression of the stromelysin 3 (ST3) gene, a new member of the matrix metalloproteinase gene family, 111 head and neck squamous cell carcinomas and 21 metastatic lymph nodes were analyzed by Northern blot. ST3 gene expression was observed in 106 carcinomas and 19 metastatic nodes, but in only 2 of 60 samples of corresponding normal tissue tested in parallel. ST3 RNA, by in situ hybridization, and ST3 protein, by immunohistochemical analysis, were specifically detected in fibroblastic cells immediately surrounding invasive cancer cells. This fibroblastic expression of the ST3 gene is characteristic among the matrix metalloproteinase genes known to be overexpressed in head and neck carcinomas, since stromelysin 2 transcripts were specifically detected in neoplastic cells, and type I collagenase transcripts in both neoplastic cells and stromal fibroblasts. Furthermore, there was a highly significant positive correlation (P < 0.0001) between ST3 RNA levels and local invasiveness by the cancer cells, suggesting that enhanced expression of the ST3 gene may contribute to the neoplastic phenotype in head and neck carcinomas.

The human spasmolytic protein, SML1/hSP, an inhibitor of spasmolytic activity and gastric acid secretion in the pig, has been shown to exhibit homology to the pS2 protein, an estrogen-dependent breast cancer marker. Moreover, SML1/hSP and pS2 are expressed at the same localization in the normal stomach and during healing of the gastrointestinal tract. Here we report the chromosomal localization, obtained by in situ hybridization, of the hSP gene (SML1) to chromosome 21 at 21q22.3. Using pulsed-field gel electrophoresis, we found SML1 to be within 230 kb of the BCEI/pS2 gene.

A total of 2500 individuals, aged 14-70 years and living in Castille and Leon (Spain), were surveyed in the spring of 1989 with regard to their drug consumption and patterns of use. 'Lifetime' drug users' rates were 30.0% for cannabis, 5.4% for cocaine, 5.2% for amphetamines, 2.4% for psychedelic drugs, 1.5% for opiates, 1.1% for tranquillizers, and 0.4% for inhalants. 'Regular' drug users were more common among cannabis--2.8% of the surveyed--and less frequent among cocaine, opiates and amphetamine users--0.3% of everyone of the substances mentioned above. 'Lifetime' drug users were more common among males than among females, among singles than among married, in those younger (18-29 years of age) and with work problems. The starting age of drug consumption ranged between the average of 15.6 years of age for those who consumed inhalants and 19.5 for those who consumed cocaine. The results allow a better understanding of the pattern of drug use in Castille and Leon (Spain), as well as the 'high' prevalence of drug use in our region.

The human stromelysin 3 (STMY3) gene, a new member of the matrix metalloproteinase (MMP) gene family, may contribute to breast cancer cell invasion, and has been localized by in situ hybridization to the long arm of chromosome 22. As demonstrated using a panel of somatic cell hybrids, the STMY3 gene is in band 22q11.2, in close proximity to the BCR gene involved in chronic myeloid leukemia, but far from the (11;22) translocation breakpoint observed in Ewing sarcoma. This position differs from that reported on chromosomes 11 and 16 for the other MMP genes, suggesting that stromelysin 3 could be a member of a new MMP subfamily.

We have analysed patterns of alcohol and regular drug consumption by Spanish drivers. Six hundred and seventy five properly completed questionnaires were received from drivers attending three medical traffic centres in Valladolid (Spain) for medical examination prior to obtaining or renewing their driving licence in 1990. Among those surveyed, 24% were 'daily' drinkers and 56.7% were 'weekly' drinkers, the majority (55.5%) being 'light' drinkers (1-39 g/day of pure alcohol). Of those surveyed 28.9% took drugs. The most commonly consumed drugs were analgesics (6.5%), anti-allergic drugs (5.2%) and oral contraceptives (4.6%). Of those drivers taking drugs 28.2% were 'daily' drinkers and 53.8% were 'weekly' drinkers. The study indicates widespread consumption of alcohol and drugs by Spanish drivers.

The human pS2 gene, isolated from the breast carcinoma cell line MCF-7 and shown to be under estrogen transcriptional control in a subclass of breast cancer cells was reported to be secreted in normal stomach surface epithelial cells, whereas additional gastrointestinal tissues like pancreas and colon do not secrete pS2 at all. In porcine pancreas, a spasmolytic polypeptide (sharing domains of homology with pS2) was observed; a corresponding human gene (hSP) was shown to be active in normal stomach mucosa. hSP and pS2 gene activity in normal and neoplastic pancreas tissues was then compared. Whereas both genes are inactive in normal pancreatic cells, activation of the pS2 sequence in a primary pancreatic carcinoma cell culture and in 23 tumor tissues was noted when investigated by immunostaining. In all cases when pS2 showed a regular 0.6 kb transcript, hSP displayed a transcript of 0.7 kb. Six of these tumors showed a reduced pS2 immunoreactivity and, at the same time, aberrant pS2 mRNA bands and a complete shut-down of the hSP gene were noted. In one case, whereas normal pancreas remained negative, the corresponding tumor and its metastasis displayed regular transcripts of pS2 and hSP. This remarkably high correlation suggests that pS2 and hSP expression in the pancreatic tumors, but not in their corresponding healthy tissue is significantly linked to molecular steps leading to tumorigenesis.

We have analysed regular drug consumption by Spanish drivers. 675 properly completed questionnaires were received from drivers attending three medical traffic centres in Valladolid (Spain) for medical examination prior to obtaining or renewing of their traffic licence in 1990. Among those surveyed, 28.9% were taking regularly drugs, mainly analgesics (6.5%), antiallergic drugs (5.2%), oral contraceptives (4.6%), anti-inflammatory drugs (4.1%), antihypertensive drugs (3.6%), tranquillizers (3.4%), hypnotics (2.8%) and drugs for rheumatic disorders (2.7%). The study indicates the frequent consumption of drugs by Spanish drivers, and suggests the need to regulate drug consumption by drivers.

The recently discovered pS2 protein is expressed under estrogen control in a subset of estrogen receptor-positive breast cancers and in an estrogen-independent manner in normal stomach mucosa. The pS2 gene belongs to a family of genes encoding peptides that contain a conserved 5-cysteine domain, the P domains. Although the function of the pS2 protein is unknown, it has been suggested that it may have cell growth stimulatory activity. We report here that expression of the pS2 gene in the digestive tract, which is normally restricted to the stomach, is strongly induced by mucosal ulcerations elsewhere in the tract, most notably in Crohn's disease. pS2 gene expression is restricted to the mucosal layers adjacent to the ulcerations, in a region where a novel epidermal growth factor-secreting cell lineage was shown to be induced by mucosal ulceration. The human hSP gene, which contains a tandem duplication of the pS2 gene P domain and is coexpressed with the pS2 gene in normal stomach mucosa but not in breast cancers, is also expressed in Crohn's disease. We suggest that pS2 gene expression may provide a useful marker for mucosal ulcerations of the digestive tract.

Seventy-two patients with advanced breast carcinoma (42% bone, 25% visceral, 5.5% soft tissue, and 27.5% multiple site metastases) were evaluated to determine the relationship between tumor expression of the estrogen-regulated protein pS2, estrogen receptor (ER) or progesterone receptor (PgR) content, and response to hormonal therapy. Twenty-nine % of tumors were pS2 positive, 64% were ER positive, and 29% were PgR positive. Of the ER-positive patients (n = 43), 15 (35%) had greater than 10% of the invasive carcinoma which immunostained for pS2 (these were considered pS2 positive). Only 3 of 24 ER-negative tumors were pS2 positive. A weak association between pS2 expression and ER content (P = 0.08) but not PgR content was observed. Of pS2-positive patients, 52% had a partial or complete response to hormonal therapy. In 24% of pS2-positive patients the disease stabilized with treatment. In contrast, 27% of pS2-negative patients had a partial or complete response. In 10% of these patients the disease stabilized. Similar associations between therapeutic response and ER or PgR were not observed. The odds of having a clinical response to hormonal therapy was greater for pS2-positive than for ER- or PgR-positive tumors. pS2 expression may define a subset of ER-positive tumors that are more likely to respond to hormonal treatment.

Expression of the pancreatic spasmolytic peptide (hSP) gene and pS2 (a gene isolated from oestrogen-induced breast carcinoma cells) were analysed in 36 samples of human stomach carcinoma. 17 tumours were investigated at the RNA level (by northern blots) as well as at the gene product level (by immunochemistry). Since pS2 had been shown to be expressed in normal stomach mucosa its activity in carcinoma samples was expected. Surprisingly, strong pS2 immunoreactivity was noted in the diffuse carcinoma type, whereas the intestinal type displayed weak reactivity. The tumour samples showing strong immunostaining expressed the regular 0.6 kb pS2 RNA band and weak staining was paralleled by aberrant transcripts. Additionally, only in tumour samples with regular pS2 transcription was the typical 0.7 kb hSP RNA band seen; samples with aberrant pS2 bands did not express hSP at all. This is the first demonstration of hSP gene activity in a human tumour.

The pS2 gene encodes for a small cysteine-rich protein, and was originally found by differential screening of a cDNA library from the human breast carcinoma cell line, MCF-7. The presence of pS2 is closely correlated with oestrogen dependence in breast carcinomas. While the function of pS2 is unknown, pS2 protein has been shown to be homologous with the gastrointestinal peptide hormone pancreatic spasmolytic polypeptide (PSP) and its human counterpart hSP, in which a 5-cysteine domain is tandemly repeated. The 5' flanking region of the pS2 gene contains an enhancer region responsive to oestrogens and to epidermal growth factor (EGF/URO). We now report that pS2 and hSP expression occurs in a wide range of endodermally-derived tissues, including the duodenum, the pancreas, and in a recently defined cell lineage associated with chronic gastrointestinal ulceration. In each case, this expression was associated with secretion of immunoreactive EGF/URO. We further show that the co-expression of pS2 and hSP in gastric surface epithelial cells is also associated with the secretion of EGF/URO in the subjacent mucous neck cells. Our results indicate that local EGF/URO secretion induces pS2 and hSP in adjacent cells, and that these molecules are then available to participate in pathophysiological responses. The finding of similar patterns of EGF/URO, hSP and pS2 expression in association with chronic damage suggests that this is a fundamental response in the healing of these tissues.

Approximately 50% of human breast tumors secrete a small cysteine-rich protein called pS2. In the human breast cancer cell line MCF-7, expression of the pS2 protein is strongly induced by estrogen, and cloning and sequence analysis of the pS2 gene has revealed an "estrogen responsive element" in the gene's 5'-flanking region. The results of immunohistochemical assays and radioimmunoassays on breast cancer biopsies indicate that the pS2 protein is a marker for hormone-dependent breast tumors and that its expression is associated with longer overall, and disease-free, survival. The pS2 protein is also expressed in normal stomach mucosa and in regenerative tissues in ulcerative diseases of the gastrointestinal tract. Its physiological function is unknown.

Application of systemic adjuvant therapy for primary breast cancer patients requires a more accurate identification of patients at high risk for recurrence. We have quantitatively assessed the cytosolic levels of estrogen-regulated pS2 protein in tumors of 205 breast cancer patients (median follow-up, 47 mo). There were no significant associations between the level of pS2 protein and tumor size, lymph node status, and differentiation grade. Using length of relapse-free survival (RFS) and overall survival (OS) as end points, 11 ng of pS2 protein/mg of cytosol protein were found as the best cutoff level to discriminate between positive (pS2+) and negative (pS2-). Patients with pS2- tumors showed significantly shorter RFS and OS (P less than 0.0001) than patients with pS2+ tumors. Also after adjustment for tumor size, lymph node status, and estrogen receptor (ER) status, pS2 negativity was associated with earlier recurrence and death. Tumors positive for pS2 (55 of 205, 27%) were almost exclusively confined to the subclass of ER+ tumors (53 of 55, 96%). The death rate for patients with pS2+ tumors was one-tenth of the death rate for patients with pS2-/ER- tumors. In the patients with ER+ tumors, the prognostic power of the pS2 status was especially present in patients whose tumors were also positive for the progesterone receptor (5-yr RFS and OS, 85% and 97% for ER+/PgR+/pS2+ tumors compared with 50% and 54% for the patients with ER+/PgR+/pS2- tumors). In patients with axillary lymph node involvement (N+), pS2 status could discriminate strongly between a good and bad prognosis group (5-yr RFS and OS, 65% and 88% for N+/pS2+ compared with 32% and 34% for N+/pS2-). A similar phenomenon was observed in patients without axillary lymph node involvement (5-yr RFS and OS, 89% and 95% for N0/pS2+ compared with 58% and 82% for N0/pS2-). We conclude that the pS2 status of human primary breast tumors is an important variable for the identification of patients at high risk for recurrence and death. Knowledge of the cytosolic pS2 status appeared of particular importance to identify patients at high risk in the ER+/PgR+ subclass of tumors, and in both the N0 and N+ subclasses of patients.

Approximately 50% of human breast tumors secrete a small cysteine-rich protein, pS2, of unknown function. pS2 protein was recently found to be homologous to a porcine protein with hormonogastric activity, pancreatic spasmolytic polypeptide (PSP), in which the 5-cysteine domain present in pS2 is tandemly duplicated. We have characterized cDNA species encoding PSP and its human and mouse counterparts, hSP and mSP. We show that hSP and pS2 are separately encoded in the genome, and that the two proteins are co-expressed in normal stomach epithelium. However, expression of hSP was not detected in breast tumors. Computer analysis revealed that the pattern of conserved cysteine residues in hSP and pS2, the P domain, is present at the N termini of two other mammalian proteins, intestinal sucrase-isomaltase and lysosomal alpha-glucosidase.

The expression of the estrogen-regulated breast-cancer-associated pS2 gene was examined in 75 stomach resections taken from 45 patients. The 600-base pS2 mRNA was found in all of the 47 non-neoplastic samples at varying levels: in the histologically normal group we observed a Poisson-type distribution, whereas 79% of the tissues exhibiting dysplastic features expressed high levels of transcript. Tumour samples expressed relatively lower pS2 mRNA, with only 18% having high levels and 43% with no detectable expression. These differences were not correlated to tumour grading, stage or site. No amplification or rearrangement of the pS2 gene was found. Immunohistochemical analysis of formalin-fixed paraffin sections, using a polyclonal antibody against pS2 protein, showed specific staining of both cytoplasm and membrane of epithelial cells in the neck region of antral and body glands as well as in luminal secretions. Immunoreactivity was observed in the sub-nuclear region of foveolar cells, with specialized gland and goblet cells in atrophic gastritis being negative. Heterogeneous but strong focal cytoplasmic staining was seen in tumour cells as well as in dysplastic epithelium. Two gastric cell lines, KATO III and MKN-45, derived from poorly differentiated adenocarcinomas also expressed pS2, whereas 3 other lines from well differentiated parental tumours did not. Genomic analysis revealed a BamHI polymorphism in Kato III cells and in the non-expressing MKN-28 cells. Immunostaining to pS2 protein was also demonstrated in the cytoplasm of KATO III cells, but neither these nor any of 30 tissues examined showed any positivity with a monoclonal antibody (MAb) to estrogen receptor. Our results suggest that pS2 is normally expressed in human stomach, possibly in association with secretory activity, and becomes down-regulated during malignancy.

In this preliminary study, we have determined the receptors in normal human prostatic tissue or from patients with benign prostatic hypertrophy and patients with prostatic cancer. A single point assay using a dextran-coated-charcoal treatment of cytosol is utilized. This method permits the elimination of endogenous steroids and is also useful when specimens are too small to provide the larger number of aliquots necessary for multiple steroid receptor analyses. The results are discussed in the light of several problems inherent to this assay. We conclude that adenoma-tissue contains more receptors than neoplasic tissues for the progesterones receptors but that there is no significant difference for the estrogen-receptor and the androgen-receptors. For all the adenoma tissues and some neoplasic tissues the level of the estrogen-receptors is always lower than the one for androgen-receptor which itself is lower than the one for progesterone-receptors. We think that the expression of the results as a ratio of two levels of steroid-receptors gives a better evaluation of the receptors. The ratios Progesterone-receptor Estrogen-receptor and Androgen-receptor/Estrogen-receptor are always greater than 1.5, except for some neoplasic tissues and we think that, as in breast tumors, there are perhaps two kinds of neoplastic tissues, one hormone-dependent, the other not.

PS2, a small estrogen-inducible secretory polypeptide with structural analogies to a growth factor, is produced by approximately 50% of human breast tumors. The function of PS2 is, however, unknown. To determine whether PS2 may play an autocrine role in the development of mammary tumors we constructed transgenic mice bearing fusion constructs designed to direct the expression of human PS2 in the lactating mammary gland under the control of the whey acidic protein (WAP) promoter. Mouse lines bearing the genomic PS2 gene under the control of the WAP promoter region (WAP-PS2-2) failed to express the transgene. However, mice harboring the fusion construct WAP-PS2-1, in which the PS2 coding sequence is inserted into the 5' untranslated region of the complete WAP gene, were observed to express the transgene. Expression was restricted to the secretory epithelium of the mammary gland during lactation, and PS2 protein was secreted into the milk. Nevertheless, no mammary gland dysplasia was observed, and PS2 expression had no discernable effect upon the physiology and/or development of the suckling young or the transgenic mother.

Our laboratory has reported previously the cloning of a complementary DNA termed pS2, corresponding to a messenger RNA (mRNA) whose synthesis is induced by estrogen in the human breast cancer cells MCF-7. Examination of the possible open reading frames of this complementary DNA has led to the prediction that the pS2 protein could be a secreted polypeptide of either 58 or 63 amino acids in length. Using a rabbit antiserum prepared against a synthetic peptide corresponding to the last 31 amino acids of the putative protein, we show that a protein with the expected migration during sodium dodecyl sulfate gel electrophoresis can indeed be immunoprecipitated from either the culture medium of MCF-7 cells grown in the presence of labeled amino acids or the in vitro translation products of MCF-7 poly(A) RNA enriched in pS2 mRNA. Furthermore, in vivo and in vitro differential amino acid labeling allows us to conclude that the mature pS2 protein is probably secreted as a 58 amino acid long peptide. Finally, we show that pS2 protein synthesis is induced in MCF-7 cells by estradiol and phenol red, but not by the antiestrogen tamoxifen, in keeping with our previous results demonstrating estrogen induction of pS2 mRNA synthesis.

The patterns of drug use were studied in 1886 14-30-year-olds in rural (less than 10,000 inhabitants) Spain in 1987. Cannabis was the most frequently consumed drug. For the 'past month', the frequency of drug consumption was 10.07% of the population for cannabis, and ranged between 1.01% for amphetamines and 0.11% for inhalants. Drug consumption was more frequent among males than among females. The earlier start in consumption was among inhalants (15.3 years) whereas cocaine consumers started later (20 years). 'New sensations and curiosity' was the most frequently reported reason for the initial consumption of the various drugs, whereas the 'search for pleasure and happiness', and 'to relax' (for tranquillizers) were the principal reasons given for continuing use. 'Euphoria' and 'well-being' were the principal effects felt under the influence of the different drugs. The present data show that drug consumption is common among young people in Spanish rural communities.

The hormone-dependence of some human breast cancers is well recognized. However, the molecular mechanisms responsible for the growth stimulation of these cancers by oestrogens are still poorly understood. With the hope of elucidating these mechanisms, we have recently cloned and studied the structure-function relationship of the human oestrogen and progestin receptors, and also undertaken a study aimed at characterizing genes whose expression is controlled by oestrogens in hormone-dependent breast cancers. We review here our findings concerning one of these genes and its expression products, the pS2 gene. We discuss also whether a systematic determination of pS2 gene expression in breast cancer biopsies could be useful to establish a new biochemical classification of these cancers which may be useful to improve the diagnosis of hormone-dependent cancers.

We have previously reported that pS2 mRNA expressed in cultured epithelial cells derived from a hormone-dependent breast carcinoma (MCF-7 cells) is also expressed in mucosa cells of normal human stomach. This mRNA encodes a putative 84 amino-acid-long protein, which is secreted by both cell types after elimination of a signal peptide. We report here the purification of the pS2 protein, its trypsin digestion and amino-acid sequencing. The MCF-7 cell-secreted protein is 60 amino-acid-long and its sequence is in complete agreement with that deduced from the mRNA sequence. The presence of an N-terminal glutamic acid indicates that the signal peptidase releases a 24 amino-acid-long signal peptide. Analysis of tryptic peptides derived from the secreted gastric pS2 protein indicates that the signal peptide and the sequence of the first 48 amino-acids are identical to those of secreted MCF-7 pS2 protein, although the N-terminal amino-acid of the gastric protein may be cyclized as a pyroglumatic acid.

The expression of the pS2 gene, which is induced by estrogen in the breast cancer cell line MCF-7, has been investigated in breast cancers by using pS2 mRNA determination in tumor specimens and immunocytochemistry to identify pS2 protein in paraffin-embedded sections. Using these assays we show that determination of pS2 gene expression allows the definition of subclasses of estrogen-receptor-containing breast cancers that may be used to more precisely identify estrogen-dependent tumors. Tumor specimens have also been analyzed for the presence of mRNAs for the estrogen receptor and for the ERBB2 oncogene. No evidence for the presence of truncated forms of estrogen-receptor mRNA has been found, and overexpression of the ERBB2 oncogene did not correlate with the steroid receptor status or pS2 gene expression.