Antonei Benjamin Csóka

The six hyaluronidase-like genes in the human and mouse genomes.

The human genome contains six hyaluronidase-like genes. Three genes (HYAL1, HYAL2 and HYAL3) are clustered on chromosome 3p21.3, and another two genes (HYAL4 and PH-20/SPAM1) and one expressed pseudogene (HYALP1) are similarly clustered on chromosome 7q31.3. The extensive homology between the different hyaluronidase genes suggests ancient gene duplication, followed by en masse block duplication, events that occurred before the emergence of modern mammals. Very recently we have found that the mouse genome also has six hyaluronidase-like genes that are also grouped into two clusters of three, in regions syntenic with the human genome. Surprisingly, the mouse ortholog of HYALP1 does not contain any mutations, and unlike its human counterpart may actually encode an active enzyme. Hyal-1 is the only hyaluronidase in mammalian plasma and urine, and is also found at high levels in major organs such as liver, kidney, spleen, and heart. A model is proposed suggesting that Hyal-2 and Hyal-1 are the major mammalian hyaluronidases in somatic tissues, and that they act in concert to degrade high molecular weight hyaluronan to the tetrasaccharide. Twenty-kDa hyaluronan fragments are generated at the cell surface in unique endocytic vesicles resulting from digestion by the glycosylphosphatidyl-inositol-anchored Hyal-2, transported intracellularly by an unknown process, and then further digested by Hyal-1. The two beta-exoglycosidases, beta-glucuronidase and beta-N-acetyl glucosaminidase, remove sugars from reducing termini of hyaluronan oligomers, and supplement the hyaluronidases in the catabolism of hyaluronan.

Hyaluronidase reduces human breast cancer xenografts in SCID mice.

A hyaluronan‐rich environment often correlate with tumor progression. and may be one mechanism for the invasive behavior of malignancies. Eradication of hyaluronan by hyaluronidase administration could reduce tumor aggressiveness and would provide, therefore, a new anti‐cancer strategy. Hyaluronan interaction with its CD44 receptor and the resulting signal transduction events may be among the mechanisms for hyaluronan‐associated cancer progression. We have shown previously that hyaluronidase treatment of breast cancer cells in vitro not only eradicates hyaluronan but also modifies expression of CD44 variant exons of tumor cells. We now determine if such effects occur in vivo and if it is accompanied by tumor regression. SCID mice bearing xenografts of human breast carcinomas were given intravenous hyaluronidase. Tumor volumes decreased 50% in 4 days. Tumor sections showed decreased hyaluronan. Intensity of staining for CD44s was not affected, whereas staining for specific CD44 variant exon isoforms was greatly reduced in residual tumors. Necrosis was not evident. Hyaluronidase, used previously as an adjunct in cancer treatment, presumably to enhance penetration of chemotherapeutic drugs, may itself have intrinsic anti‐cancer activity. Removing peritumor hyaluronan appears to cause an irreversible change in tumor metabolism. Continuous hyaluronan binding to CD44 variant exon isoforms may also be required to stabilize inherently unstable isoforms that participate perhaps in tumor progression. Further investigation is required to confirm a cause and effect relationship between loss of hyaluronan, changes in CD44 variant exon expression and tumor reduction. If confirmed, hyaluronidase may provide a new class of anti‐cancer therapeutics and one without toxic side effects.

HYAL1(LUCA-1), a candidate tumor suppressor gene on chromosome 3p21.3, is inactivated in head and neck squamous cell carcinomas by aberrant splicing of pre-mRNA

The hyaluronidase first isolated from human plasma, Hyal-1, is expressed in many somatic tissues. The Hyal-1 gene, HYAL1, also known as LUCA-1, maps to chromosome 3p21.3 within a candidate tumor suppressor gene locus defined by homozygous deletions and by functional tumor suppressor activity. Hemizygosity in this region occurs in many malignancies, including squamous cell carcinomas of the head and neck. We have investigated whether cell lines derived from such malignancies expressed Hyal-1 activity, using normal human keratinocytes as controls. Hyal-1 enzyme activity and protein were absent or markedly reduced in six of seven carcinoma cell lines examined. Comparative genomic and fluorescence in situ hybridization identified chromosomal deletions of one allele of HYAL1 in six of seven cell lines. Initial RT–PCR analyses demonstrated marked discrepancies between levels of HYAL1 mRNA and protein. Despite repeated sequence analyses, no mutations were found. However, two species of transcripts were identified when primers were used that included the 5′ untranslated region. The predominant mRNA species did not correlate with protein translation and contained a retained intron. A second spliced form lacking this intron was found only in cell lines that produced Hyal-1 protein. Inactivation of HYAL1 in these tumor lines is a result of incomplete splicing of its pre-mRNA that appears to be epigenetic in nature.