Eva Forgacs

Stromal and Epithelial Expression of Tumor Markers Hyaluronic Acid and HYAL1 Hyaluronidase in Prostate Cancer

Hyaluronic acid (HA), a glycosaminoglycan, regulates cell adhesion and migration. Hyaluronidase (HAase), an endoglycosidase, degrades HA into small angiogenic fragments. Using an enzyme-linked immunosorbent assay-like assay, we found increased HA levels (3–8-fold) in prostate cancer (CaP) tissues when compared with normal (NAP) and benign (BPH) tissues. The majority (∼75–80%) of HA in prostate tissues was found to exist in the free form. Primary CaP fibroblast and epithelial cells secreted 3–8-fold more HA than respective NAP and BPH cultures. Only CaP epithelial cells and established CaP lines secreted HAase and the secretion increased with tumor grade and metastasis. The pH activity profile and optimum (4.2; range 4.0–4.3) of CaP HAase was identical to the HYAL1-type HAase present in human serum and urine. Full-length HYAL1 transcript and splice variants were detected in CaP cells by reverse transcriptase-polymerase chain reaction, cloning, and sequencing. Immunoblotting confirmed secretion of a ∼60-kDa HYAL1-related protein by CaP cells. Immunohistochemistry showed minimal HA and HYAL1 staining in NAP and BPH tissues. However, a stromal and epithelial pattern of HA and HYAL1 expression was observed in CaP tissues. While high HA staining was observed in tumor-associated stroma, HYAL1 staining in tumor cells increased with tumor grade and metastasis. The gel-filtration column profiles of HA species in NAP, BPH, and CaP tissues were different. While the higher molecular mass and intermediate size HA was found in all tissues, the HA fragments were found only in CaP tissues. In particular, the high-grade CaP tissues, which showed both elevated HA and HYAL1 levels, contained angiogenic HA fragments. The stromal-epithelial HA and HYAL1 expression may promote angiogenesis in CaP and may serve as prognostic markers for CaP.

Mutation analysis of the PTEN/MMAC1 gene in lung cancer

We studied PTEN/MMAC1, a newly discovered candidate tumor suppressor gene at 10q23.3, for mutations in lung cancer. One hundred and thirty-six lung cancer cell line DNAs (66 small cell lung cancers, SCLC, 61 non-small cell lung cancers, NSCLC, four mesotheliomas, five extrapulmonary small cell cancers) were analysed for PTEN/MMAC1 homozygous deletions and five (8%) SCLC lines showed homozygous deletions interrupting the PTEN/MMAC1 gene. Using single stranded conformation polymorphism (SSCP) analysis, we screened the PTEN/MMAC1 open reading frame of 53 lung cancer cell line cDNAs for point mutations and found that 3/35 SCLCs and 3/18 NSCLCs contained homozygous amino acid sequence altering mutations. Northern blot analysis revealed that expression of the PTEN/MMAC1 gene was considerably lower in all the tumor cell lines with point mutations while no expression was detected for cell lines with PTEN/MMAC1 homozygous deletions. Mutation analysis of 22 uncultured, microdissected, primary SCLC tumors and metastases showed two silent mutations, and two apparent homozygous deletions. We also discovered a processed pseudogene (PTEN2) which has 98.5% nt identity to PTEN/MMAC1, that needs to be accounted for in cDNA mutation analysis. Our findings suggest that genetic abnormalities of the PTEN/MMAC1 gene are only involved in a relatively small subset of lung cancers.

Repeat Polymorphisms within Gene Regions: Phenotypic and Evolutionary Implications

We have developed an algorithm that predicted 11,265 potentially polymorphic tandem repeats within transcribed sequences. We estimate that 22% (2,207/9,717) of the annotated clusters within UniGene contain at least one potentially polymorphic locus. Our predictions were tested by allelotyping a panel of approximately 30 individuals for 5% of these regions, confirming polymorphism for more than half the loci tested. Our study indicates that tandem-repeat polymorphisms in genes are more common than is generally believed. Approximately 8% of these loci are within coding sequences and, if polymorphic, would result in frameshifts. Our catalogue of putative polymorphic repeats within transcribed sequences comprises a large set of potentially phenotypic or disease-causing loci. In addition, from the anomalous character of the repetitive sequences within unannotated clusters, we also conclude that the UniGene cluster count substantially overestimates the number of genes in the human genome. We hypothesize that polymorphisms in repeated sequences occur with some baseline distribution, on the basis of repeat homogeneity, size, and sequence composition, and that deviations from that distribution are indicative of the nature of selection pressure at that locus. We find evidence of selective maintenance of the ability of some genes to respond very rapidly, perhaps even on intragenerational timescales, to fluctuating selective pressures.

Molecular genetic abnormalities in the pathogenesis of human lung cancer

In the past few years our knowledge of the molecular pathogenesis of lung cancer has significantly increased. There are several molecular mechanisms involved in the multistage carcinogenesis through which respiratory epithelial cells become preneoplastic and then invasive cancer. In this review we summarize some of these changes including, genomic alterations such as loss of heterozygosity and microsatellite alterations, autocrine-paracrine loops, alterations in oncogenes and tumor suppressor genes, tumor angiogenesis, aberrant promoter methylation and inherited predisposition to lung cancer. Translation of these findings to the clinic is also discussed.

Structural basis for drug-induced allosteric changes to human β-cardiac myosin motor activity

Omecamtiv Mecarbil (OM) is a small molecule allosteric effector of cardiac myosin that is in clinical trials for treatment of systolic heart failure. A detailed kinetic analysis of cardiac myosin has shown that the drug accelerates phosphate release by shifting the equilibrium of the hydrolysis step towards products, leading to a faster transition from weak to strong actin-bound states. The structure of the human β-cardiac motor domain (cMD) with OM bound reveals a single OM-binding site nestled in a narrow cleft separating two domains of the human cMD where it interacts with the key residues that couple lever arm movement to the nucleotide state. In addition, OM induces allosteric changes in three strands of the β-sheet that provides the communication link between the actin-binding interface and the nucleotide pocket. The OM-binding interactions and allosteric changes form the structural basis for the kinetic and mechanical tuning of cardiac myosin.

Searching for microsatellite mutations in coding regions in lung, breast, ovarian and colorectal cancers

RepX represents a new informatics approach to probe the UniGene database for potentially polymorphic repeat sequences in the open reading frame (ORF) of genes, 56% of which were found to be actually polymorphic. We now have performed mutational analysis of 17 such sites in genes not found to be polymorphic (<0.03 frequency) in a large panel of human cancer genomic DNAs derived from 31 lung, 21 breast, seven ovarian, 21 (13 microsatellite instability (MSI)+ and eight MSI−) colorectal cancer cell lines. In the lung, breast and ovarian tumor DNAs we found no mutations (<0.03–0.04 rate of tumor associated open reading frame mutations) in these sequences. By contrast, 18 MSI+ colorectal cancers (13 cancer cell lines and five primary tumors) with mismatch repair defects exhibited six mutations in three of the 17 genes (SREBP-2, TAN-1, GR6) (P<0.000003 compared to all other cancers tested). We conclude that coding region microsatellite alterations are rare in lung, breast, ovarian carcinomas and MSI (−) colorectal cancers, but are relatively frequent in MSI (+) colorectal cancers with mismatch repair deficits.

The kinetic mechanism of mouse myosin VIIA.

Myosin VIIa is crucial in hearing and visual processes. We examined the kinetic and association properties of the baculovirus expressed, truncated mouse myosin VIIa construct containing the head, all 5IQ motifs and the putative coiled coil domain (myosin VIIa-5IQ). The construct appears to be monomeric as determined by analytical ultracentrifugation experiments, and only single headed molecules were detected by negative stain electron microscopy. The relatively high basal steady-state rate of 0.18 s−1 is activated by actin only by ∼3.5-fold resulting in a Vmax of 0.7 s−1 and a KATPase of 11.5 μm. There is no single rate-limiting step of the ATP hydrolysis cycle. The ATP hydrolysis step (M·T ⇄ M·D·P) is slow (12 s−1) and the equilibrium constant (KH) of 1 suggests significant reversal of hydrolysis. In the presence of actin ADP dissociates with a rate constant of 1.2 s−1. Phosphate dissociation is relatively fast (>12 s−1), but the maximal rate could not be experimentally obtained at actin concentrations ≤ 50 μm because of the weak binding of the myosin VIIa-ADP-Pi complex to actin. At higher actin concentrations the rate of attached hydrolysis (0.4 s−1) becomes significant and partially rate-limiting. Our findings suggest that the myosin VIIa is a “slow”, monomeric molecular motor with a duty ratio of 0.6.