GLI3 is a transcriptional effector of the developmentally important hedgehog (Hh) signaling pathway. Here we report the production of mouse monoclonal antibody (MAb) against putative repressive motif ...in GLI3 (GLI3pRM). BALB/c mice were immunized with purified recombinant human GLI3pRM protein, and the splenocytes from these mice were fused with myeloma cell line (SP2/0) by using standard hybridoma production techniques. Resulting hybridomas producing anti-GLI3pRM antibodies were screened by enzyme-linked immunosorbent assay (ELISA) and isotyped. The specificity of MAb 5E1 was determined based on its activity in Western blot and immunofluorescence analyses of the human NT2/D1 cell line. The results showed that MAb 5E1 was immunoglobulin IgM/kappa, recognizing recombinant human GLI3pRM specifically. In addition, MAb 5E1 bound to the full-length (FL-GLI3) as well as a short protein (GLI3R) and did not cross-react with a similar region in GLI2. MAb 5E1 could also be used to detect the expression of Gli3 in mouse cell lines and embryonic tissues.
This thesis focusses on the role of hyaluronan receptor CD44 in tumor progression and metastasis of a well-characterized mouse fibrosarcoma/tumor metastasis model system. First, CD44 levels and ...function were characterized in relation to oncogenic transformation and metastatic capacity. In 10% serum, CD44s level and its ability to bind HA were low in 3T3 cells and in nonmetastatic sis transformants; those parameters were upregulated in metastatic ras transformants. In 5% serum, 3T3 cells expressed high levels of CD44s capable of binding hyaluronan irrespective of oncogenic transformation, suggesting that ras makes CD44 levels serum-independent. PDGF is the major CD44-regulating factor in serum and its CD44-stimulating activity closely parallels its stimulation of chemotaxis suggesting that CD44 may be involved in this process. CD44 levels are also upregulated in confluent cultures of sis-transformed cells (irrespective of the serum concentration) in a manner partially dependent on secreted PDGF(c-sis product). To test the role of CD44s in tumor metastasis more directly, human CD44s cDNA was stably transfected into 3T3 cells and into nonmetastatic fibrosarcoma cells: HUSI (sis-transformed 3T3 cells) and IIIA4 (ras-revertant from late-passage Ki-ras transformants). Tumor development by transfectants was analysed in nude mice at the subcutaneous site. Human CD44s over-expression transforms 3T3 cells in select cases and induces full metastatic progression. The analysis of tumors derived from hCD44s-expressing 3T3 cells revealed complex regulation of hCD44s in tumor progression: down-regulation in primary tumors, re-expression in micrometastases and subsequent loss during the outgrowth of overt metastases. In our two fibrosarcoma systems, hCD44s expression caused some inhibition of tumor development but again induced full malignant progression with the same type of complex regulation of hCD44s expression as seen with nontransformed 3T3 cells. In this system, down-regulation of hCD44s in primary tumors by methylation of the hCD44s gene was demonstrated. In contrast, hCD44s gene was not methylated and hCD44s protein was present in lung micrometastatic cells. After tail vein injections, hCD44s dramatically increases retention of tumor cells in lung vasculature which may be the mechanism by which CD44s induces micrometastasis. These results suggest that CD44s is a novel type of metastasis-promoting molecule that is counter-selected during tumor outgrowth.
Truncation mutations of the
GLI3
zinc finger transcription factor can cause Greig cephalopolysyndactyly syndrome (GCPS), Pallister–Hall syndrome (PHS), and postaxial polydactyly type A (PAP-A). GLI3 ...is homologous to
Drosophila
Cubitus interruptus (Ci), which regulates the
patched
(
ptc
),
gooseberry
(
gsb
), and
decapentaplegic
(
dpp
) genes. Ci is sequestered in the cytoplasm and is subject to posttranslational processing whereby the full-length transcriptional activator form (Ci
155
) can be cleaved to a repressor form (Ci
75
). Under hedgehog signaling, the Ci
155
form translocates to the nucleus whereas in the absence of hedgehog, the Ci
75
form translocates to the nucleus. Based on the correlation of GLI3 truncation mutations and the human phenotypes, we hypothesized that GLI3 shows transcriptional activation or repression activity and subcellular localization similar to Ci. Here we show that full-length GLI3 localizes to the cytoplasm and activates
PTCH1
expression, which is similar to full-length Ci
155
. PHS mutant protein (GLI3-PHS) localizes to the nucleus and represses GLI3-activated
PTCH1
expression, which is similar to Ci
75
. The GCPS mutant protein has no effect on GLI3-activated
PTCH1
transcription, consistent with the role of haploinsufficiency in this disorder. The PAP-A mutant protein (GLI3-PAP-A) showed less specific subcellular localization but still inhibited GLI3-activated
PTCH1
transcription, suggesting it may be a weaker allele than the GLI3-PHS mutation. These data show that
GLI3
mutations in humans mimic functional effects of the
Drosophila ci
gene and correlate with the distinct effects of these mutations on human development.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Human CD44 standard isoform (hCD44s) cDNA regulated by a high-expressing promoter was transfected into Balb/c 3T3 cells and the tumorigenic and metastatic capacities of the transfectants were ...investigated in nude mice at the subcutaneous site. One of three transfectants was tumorigenic. hCD44s expression was lost in the cells of large primary tumors using this tumorigenic clone. These tumors were extremely aggressive giving overt metastases and micrometastases to several sites including mesentery, stomach, liver, diaphragm, pancreas and lung. Micrometastatic cells re-expressed hCD44s, consistent with its importance for early steps in the metastatic cascade. hCD44s was not expressed in overt metastases; most probably the expression was lost during the outgrowth of micrometastases into overt metastatic tumors. Thus hCD44s expression in murine 3T3 cells does induce tumorigenicity in select cases, is not compatible with aggressive outgrowth of primary or secondary tumors, and is advantageous for early steps in metastatic spread. These results suggest that CD44s is an example of a novel type of 'metastasis' molecule that is disadvantageous for tumor growth and is only transiently advantageous during metastatic spreading of tumor cells to distant organs.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Oncogene-dependent regulation and tumor relatedness of CD44 expression were investigated in Balb/c 3T3 cells and their derivatives transformed with different ras oncogenes (metastatic tumor model) or ...the human c-sis oncogene (non-metastatic model). Ras transformants using either the Harvey or Kirsten oncogenes expressed high levels of cell surface CD44 protein that bound fluoresceinated hyaluronan (HA). Much lower levels of CD44 were expressed in parental 3T3 cells, ras- revertants generated from Kirsten-transformed cells, or c-sis transformants, confirming the significance of the ras oncogene in this upregulation. To determine whether endogenous HA regulates these parameters, hyaluronidase treatment of ras transformants exposed more cell surface CD44 to anti-CD44 antibody and increased fluoresceinated HA binding; this did not occur with 3T3 or c-sis transformants. CD44 expression and its HA-binding function were conserved in a panel of in vivo primary and lung metastatic tumor cell lines derived from ras transformants. Ras transformants also retained the ability to downregulate CD44 protein levels in confluent cultures which occurred through a translational or post-translational mechanism (as CD44 mRNA levels were not reduced). These results taken together demonstrate that ras-dependent regulation of CD44 may correlate with tumor progression and metastasis in vivo, possibly (although not exclusively) supporting CD44's importance in metastatic progression.
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