Genetic engineering of human cytokines. Cloning, structure and expression of human interferons-#alpha# and interleukin-2

Summary in Latvian, English, RussianAvailable from Latvian Academic Library / LAL - Latvian Academic LibrarySIGLELVLatvi

HYAL1 and HYAL2 inhibit tumour growth in vivo but not in vitro.

BACKGROUND: We identified two 3p21.3 regions (LUCA and AP20) as most frequently affected in lung, breast and other carcinomas and reported their fine physical and gene maps. It is becoming increasingly clear that each of these two regions contains several TSGs. Until now TSGs which were isolated from AP20 and LUCA regions (e.g.G21/NPRL2, RASSF1A, RASSF1C, SEMA3B, SEMA3F, RBSP3) were shown to inhibit tumour cell growth both in vitro and in vivo. METHODOLOGY/PRINCIPAL FINDINGS: The effect of expression HYAL1 and HYAL2 was studied by colony formation inhibition, growth curve and cell proliferation tests in vitro and tumour growth assay in vivo. Very modest growth inhibition was detected in vitro in U2020 lung and KRC/Y renal carcinoma cell lines. In the in vivo experiment stably transfected KRC/Y cells expressing HYAL1 or HYAL2 were inoculated into SCID mice (10 and 12 mice respectively). Tumours grew in eight mice inoculated with HYAL1. Ectopic HYAL1 was deleted in all of them. HYAL2 was inoculated into 12 mice and only four tumours were obtained. In 3 of them the gene was deleted. In one tumour it was present but not expressed. As expected for tumour suppressor genes HYAL1 and HYAL2 were down-expressed in 15 fresh lung squamous cell carcinomas (100%) and clear cell RCC tumours (60-67%). CONCLUSIONS/SIGNIFICANCE: The results suggest that the expression of either gene has led to inhibition of tumour growth in vivo without noticeable effect on growth in vitro. HYAL1 and HYAL2 thus differ in this aspect from other tumour suppressors like P53 or RASSF1A that inhibit growth both in vitro and in vivo. Targeting the microenvironment of cancer cells is one of the most promising venues of cancer therapeutics. As major hyaluronidases in human cells, HYAL1 and HYAL2 may control intercellular interactions and microenvironment of tumour cells providing excellent targets for cancer treatment

Effect of <i>HYAL1</i> and <i>HYAL2</i> transgenes on colony formation efficiency in KRC/Y and U2020 cells compared to empty pETE vector (negative control) and wild type or mutated <i>FUS1</i> transgenes.

<p>Graphical representation summarizing three independent experiments and photographic images of Petri dishes stained with methylene blue. Values are the mean ±s.d. of three separate experiments each calculated from triplicate plates.</p

QPCR mRNA expression profile of <i>HYAL1</i> and <i>HYAL2</i> in SCC and RCC biopsies.

<p>The Y axis indicates the values of relative expression level of target genes in log₁₀ scale in tumour samples relative to control normal samples normalized to the reference gene <i>GAPDH.</i> The X axis shows the cDNA samples isolated from tumours at different stages (I–III). Open bars show HYAL1 and hatched bars HYAL2 expression.</p

CyQUANT Cell Proliferation Assay.

<p>Effect of expression of <i>HYAL1</i> transgene in KRC/Y (A) and in U2020 (B) cells. The same is shown for <i>HYAL2</i> (KRC/Y in C and U2020 in D). Experiments were done in triplicates in the absence of doxycycline. The same experiments were done in the presence of doxycycline and showed similar results (data not shown). Plotted data points represent averages of triplicate samples, the plotted line is a linear regression fit of all data points. The assay is designed to produce a linear analytical response from at least 100–20,000 cells per well in most cell lines.</p

Analysis of <i>HYAL1</i> and <i>HYAL2</i> stably transformed KRC/Y clones.

<p>Northern analysis (A) of tetracycline regulated clones. (+), tetracycline (Tet) or doxycycline is present, gene is OFF. (−), tetracycline or doxycycline is absent, gene is ON. Growth inhibition of KRC/Y cells with <i>HYAL1</i> (B) and <i>HYAL2</i> (C) transgenes <i>in vitro</i>. Tumour growth inhibition of KRC/Y cells by <i>HYAL1</i> and <i>HYAL2 in vivo</i> in SCID mice (D). Mice were drinking water with tetracycline (+Tet, gene is OFF) or without (−Tet, gene is ON) but for simplicity curves are shown only for mice when genes were ON (no tetracycline).</p