Overexpression of cyclin D mRNA distinguishes invasive and in situ breast carcinomas from non-malignant lesions.

The elucidation of molecular alterations that occur during human breast cancer progression may contribute to the development of preventative strategies. Using in situ hybridizations on a cohort of 94 biopsy lesions, quantitatively increased cyclin D mRNA expression levels were observed in only 18% of benign lesions, which confer no or slightly increased breast cancer risk, and 18% of premalignant atypical ductal hyperplasias, which confer a four to fivefold increase in breast cancer risk. The transition to carcinoma was accompanied by frequent cyclin D mRNA overexpression in 76% of low-grade ductal carcinomas in situ, 87% of higher grade comedo ductal carcinomas in situ and 83% of infiltrating ductal breast carcinomas. The data identify a molecular event that may separate benign and premalignant human breast lesions from any form of breast carcinoma.

Transfection of thrombospondin 1 complementary DNA into a human breast carcinoma cell line reduces primary tumor growth, metastatic potential, and angiogenesis.

Previous studies demonstrated that metastatic MDA-MB-435 breast carcinoma cells synthesized and secreted less of the extracellular matrix protein thrombospondin 1 (TSP1) than nonmetastatic breast carcinoma cell lines, a trend also observed for melanoma and lung carcinoma cell lines. To directly examine the effect of tumor cell TSP1 expression on tumor growth and metastasis. MDA-MB-435 cells were transfected with full length THBS-1 cDNA linked to a constitutive cytomegalovirus promoter, or with the cytomegalovirus vector alone. Injection of transfected clones that overexpressed TSP1 into the mammary fat pad of nude mice resulted in a dose-dependent inhibition of primary tumor size and an inhibition of spontaneous pulmonary metastases, which occurred in 21-30% of THBS-1 transfectants compared to 44-49% of controls (P = 0.007). An additional clone was identified that overexpressed a COOH-terminally truncated TSP1. This clone produced larger primary tumors and an increase in the occurrence of metastases relative to control transfectants, suggesting the participation of a previously understudied region of TSP1 in the regulation of tumor progression. The THBS-1 and control transfectants did not exhibit significant differences in growth, colonization, or motility in vitro. However, a relative reduction in capillary densities in primary tumors formed by the wild-type THBS-1 transfectants was observed, suggestive of an angiostatic effect. The data indicate that tumor cell production of TSP1 can exert a significant inhibitory effect on tumor progression in the MDA-MB-435 breast carcinoma cell line, which may be attributable in part to a reduction in angiogenesis.

Positional cloning approach to the dominant polycystic kidney disease gene, PKD1.

Positional cloning is a powerful strategy for identifying the site of disease-producing mutations when the underlying biochemical defect is unknown. The approach also offers new methods for the presymptomatic diagnosis of genetic disease. Using these methods we have localized the PKD1 gene, mutated in the majority of PKD1 families, to a small (500 kb) segment of chromosome 16, band p13.3. Virtually all of this interval has been cloned in cosmids and lambda bacteriophage. Over 20 sets of non-overlapping cDNA clones have been isolated from the region. Sequence and mutational analyses are currently underway. In addition, a set of polymorphic clones has been identified for presymptomatic diagnosis. Included in this set are several highly variable [CA]n microsatellite repeats. These highly informative markers can be rapidly assayed from a small amount of genomic DNA using the polymerase chain reaction. Despite these advances, presymptomatic diagnosis cannot be established with certainty in many families. However, identification of the PKD1 gene itself will eventually allow diagnosis by direct detection of mutations.

Angiogenesis and colonization in the tumor metastatic process: basic and applied advances.

Tumor metastasis is a major cause of death for cancer patients. This review proposes that the final steps in the development of a distant metastasis may be the most productive targets for clinical development. It cannot be guaranteed that, in "metastasis-free" patients, tumor cells have not invaded out of the primary lesion, intravasated and extravasated from the circulatory system, and are sitting at distant sites as occult micrometastases. The remaining processes involved in outgrowth at metastatic sites, colonization and angiogenesis, are reviewed. Colonization is thought to be accomplished by clonally dominant cell populations through progressive independence from exogenous growth factors, production of growth factors, and stimulatory proliferative responses to traditionally inhibitory cytokines. Therapeutic efforts aimed at interrupting the switch in tumor cell responsiveness to cytokines, rather than to any one specific cytokine, may be most successful at inhibiting metastatic colonization. Angiogenesis has been demonstrated to be directly or indirectly induced by a plethora of cytokines. Partial suppression of neovascularization can be achieved in tissue culture and animal models using various natural and pharmaceutical angiostatic agents. However, as with clonal dominance, such agents must be able to suppress the redundant effects of angiogenesis-promoting factors. This review discusses the current literature on colonization and angiogenesis, emphasizing its underlying mechanisms and potential therapeutic applications.

A transducin-like gene maps to the autosomal dominant polycystic kidney disease gene region.

A novel human gene (sazD) that maps to the autosomal dominant polycystic kidney disease region shares sequence similarity with members of the beta-transducin superfamily. The cDNA sazD-c predicts an approximately 58-kDa protein (sazD) with seven internal repeats, similar to the WD-40 motif of the transducin family. The size of this protein family has been expanding rapidly; however, neither the structure nor the function of this repeated motif is known. Preliminary data do not suggest that sazD is mutated in patients with polycystic kidney disease.

A new human gene located in the PKD1 region of chromosome 16 is a functional homologue to ERV1 of yeast.

A new human gene has been identified on chromosome 16 in the interval containing the locus for polycystic kidney disease (PKD1) by analysis of a genomic cosmid clone and cDNAs. The gene contains at least one intron and is actively transcribed in tissues from kidney and brain. The putative gene product is predicted to be homologous to the yeast scERV1 protein by virtue of the high degree of identity (42%) over the entire length of the polypeptides. In former studies the yeast scERV1 gene was found to be essential for oxidative phosphorylation, the maintenance of mitochondrial genomes, and the cell-division cycle. In this study a yeast expression vector with a chimeric reading frame coding for the first 21 amino acids of the yeast protein and the terminal 100 amino acid residues of the human factor was transformed into yeast mutants with two different defects for scERV1. The chimeric human gene product was able to complement the yeast mutants and restored near normal viability. This identifies the human gene as a structural and functional homologue of the scERV1 gene.

CpG island in the region of an autosomal dominant polycystic kidney disease locus defines the 5' end of a gene encoding a putative proton channel.

In an attempt to isolate candidate genes for autosomal dominant polycystic kidney disease, a number of CpG-rich islands have been identified from a region defined genetically as the site of disease mutations. Genomic fragments adjacent to one of these islands were used to isolate cDNAs from both HeLa cells and cultured cystic epithelium that encode a 155-amino acid peptide having four putative transmembrane domains. The corresponding transcript was found in all tissues tested but was most abundant in brain and kidney. Potential control response elements were identified in the genomic region 5' to the initiation codon. The deduced amino acid sequence has 93% similarity to the 16-kDa proteolipid component that is believed to be part of the proton channel of the vacuolar H(+)-ATPase. Possible roles for a mutated proton channel in the pathogenesis of cystic disease were considered. However, sequencing of cDNAs corresponding to both alleles of an affected individual revealed no differences in the deduced amino acid sequence. Moreover, transcript size and abundance were not altered in cystic kidney.

Cosmid walking and chromosome jumping in the region of PKD1 reveal a locus duplication and three CpG islands.

The locus responsible for the most common form of autosomal dominant polycystic kidney disease (PKD1) is located on chromosome 16p13.3. Genetic mapping studies indicate that PKD1 is flanked on the proximal side by the DNA marker 26.6 (D16S125). Here we show that 26.6 has undergone a locus duplication and that the two loci are less than 150kb apart. One of the two loci contains a polymorphic TaqI site that has been used in genetic studies and represents the proximal boundary for the PKD1 locus. We demonstrate that the polymorphic locus is the more proximal of the two 26.6-hybridizing loci. Therefore, four cosmids isolated from the distal 26.6-hybridizing locus contain candidate sequences for the PKD1 gene. These cosmids were found to contain two CpG islands that are likely markers for transcribed regions. A third CpG island was detected and cloned by directional chromosome jumping.

Fine genetic localization of the gene for autosomal dominant polycystic kidney disease (PKD1) with respect to physically mapped markers.

PKD1, the gene for the chromosome 16-linked form of autosomal dominant polycystic kidney disease, has previously been genetically mapped to an interval bounded by the polymorphic loci Fr3-42/EKMDA2 distally and O327hb/O90a proximally. More recently, 26.6PROX was identified as the closest proximal flanking locus. We set out to refine the localization of PKD1 by identifying a series of single recombinant events between the flanking markers Fr3-42/EKMDA2 and O327hb/O90a and analyzing them with a new set of polymorphic loci that have been physically mapped within the PKD1 interval. We identified 11 such crossovers in eight families; 6 of these fell into the interval between GGG1 and 26.6PROX, a distance of less than 750 kb. Three of these crossovers placed PKD1 proximal to GGG1 and two crossovers placed PKD1 distal to 26.6PROX. Both of the latter also placed PKD1 telomeric to a locus 92.6SH1.0, which lies 200-250 kb distal to 26.6PROX. The sixth recombinant, however, placed the disease mutation proximal to the locus 92.6SH1.0. Several possible explanations for these observations are discussed. An intensive study to locate deletions, insertions, and other chromosomal rearrangements associated with PKD1 mutations failed to detect any such abnormalities. Thus we have defined, in genetic and physical terms, the segment of 16p13.3 where PKD1 resides and conclude that a gene-by-gene analysis of the region will be necessary to identify the mutation(s).

Human-mouse homologies in the region of the polycystic kidney disease gene (PKD1).

Autosomal dominant polycystic kidney disease (PKD1) is linked to the alpha-globin locus near the telomere of chromosome 16p. We established the existence of a conserved linkage group in mouse by mapping conserved sequences and cDNAs from the region surrounding the PKD1 gene in the mouse genome. Results obtained with the BXD recombinant strain system and somatic cell hybrids show the homologous region to be located on mouse chromosome 17 near the globin pseudogene Hba-ps4, an unprocessed alpha-like globin gene. The markers we mapped are widely distributed over the region known to contain the PKD1 gene, and it is therefore likely that the mouse homologue of PKD1 is also located on mouse chromosome 17.

The gene for autosomal dominant polycystic kidney disease lies in a 750-kb CpG-rich region.

PKD1, the locus most commonly affected by mutations that produce autosomal dominant polycystic kidney disease (ADPKD), has previously been localized to chromosome 16p13.3. Since no cytogenetic abnormalities have been found in association with ADPKD, flanking genetic markers have been required to define an interval--the PKD1 region--that contains the PKD1 gene. In this report we demonstrate, through the construction of a long-range restriction map that links the flanking genetic markers GGG1 (D16S84) and 26.6PROX (D16S125), that the PKD1 gene lies within an extremely CpG-rich 750-kb segment of chromosome 16p13.3. Approximately 90% of this region has been cloned in three extensive cosmid/bacteriophage contigs. The cloned DNA is a valuable resource for identifying new closer flanking genetic markers and for isolating candidate genes from the region.