The wild-type protein product of the p53 tumor suppressor gene can activate transcription of genes which are involved in mediating either growth arrest, e.g. WAF1 or apoptotis, e.g. BAX and PIG3. Additionally, p53 can repress a variety of promoters, which, in turn, may be responsible for the functional activities exhibited by p53. This study shows that the Q22, S23 double mutation, which is known to inactivate a p53 transactivation subdomain located within the initial 40 residues of the protein, while abrogating transactivation from the WAF1 promoter, only attenuates apoptosis triggering, transactivation from other p53-responsive promoters and repression of promoters by p53. The Q53, S54 double mutation, which inactivates another p53 transactivation subdomain situated between amino acids 43 and 73 results in attenuation of all of the aforementioned p53 activities. In contrast to the Q22, S23 double mutation, this latter mutation set does not alter mdm-2-mediated inhibition and degradation of p53. Finally, mutation of all four residues results in complete abrogation of every p53 activity mentioned above.

Here, we report the identification of a human orthologue of fibulin-4, along with analysis of its biosynthetic processing and mRNA expression levels in normal and tumour tissues. Comparative sequence analysis of fibulin-4 cDNAs revealed apparent polymorphisms in the signal sequence that could account for previously reported inefficient secretion in fibulin-4 transfectants. In vitro translation of fibulin-4 mRNA revealed the presence of full-length and truncated polypeptides, the latter apparently generated from an alternative translation initiation site. Since this polypeptide failed to incorporate into endoplasmic reticulum membrane preparations, it was concluded that it lacked a signal sequence and thus could represent an intracellular form of fibulin-4. Using fluorescence in situ hybridisation analysis, the human fibulin-4 gene was localised to chromosome 11q13, this region being syntenic to portions of mouse chromosomes 7 and 19. Considering the fact that translocations, amplifications and other rearrangements of the 11q13 region are associated with a variety of human cancers, the expression of human fibulin-4 was evaluated in a series of colon tumours. Reverse transcription-polymerase chain reaction analysis of RNA from paired human colon tumour and adjacent normal tissue biopsies showed that a significant proportion of tumours had V V2^7-fold increases in the level of fibulin-4 mRNA expression. Taken together, results reported here suggest that an intracellular form of fibulin-4 protein may exist and that dysregulated expression of the fibulin-4 gene is associated with human colon tumourigenesis. ß

No abstract

To understand and dissect the mechanisms driving human NK cell proliferation, we exploited the methodology used in cell therapy to numerically expand NK cells in the presence of K562-derived artificial APC (aAPCs) and cytokines. For four consecutive weeks, high expression of CD137L by a K562-derived aAPC cell line could sustain NK cell expansion by 3 3 10 5 -fold, whereas low expression of CD137L by the parental K562 cell line only supported the expansion by 2 3 10 3 -fold. The level of expression of CD137L, however, did not modulate the sensitivity of K562 cells to the intrinsic cytotoxicity of NK cells. Similarly, the low NK cell proliferation in the presence of the parental K562 cell line and cytokines was increased by adding agonistic anti-CD137 Abs to levels similar to CD137L-expressing K562-derived aAPCs. Finally, synergy between IL-15 and IL-21 was observed only upon CD137 engagement and the presence of aAPCs. Therefore, we conclude that NK cell proliferation requires cell-to-cell contact, activation of the CD137 axis, and presence of IL-15 (or its membranous form) and IL-21. By analogy with the three-signal model required to activate T cells, we speculate that the cell-to-cell contact represents "signal 1," CD137 represents "signal 2," and cytokines represent "signal 3." The precise nature of signal 1 remains to be defined.

The clinical potential of the p53 tumor suppressor gene is being evaluated currently for gene therapy of cancer. We have built a variant of wild-type p53, chimeric tumor suppressor 1 (CTS1), in which we have replaced the domains that mediate its inactivation. CTS1 presents some very interesting properties: (a) enhanced transcriptional activity; (b) resistance to the inactivation by oncogenic forms of p53; (c) resistance to the inactivation by MDM2; (d) lower sensitivity to E6-induced degradation; (e) ability to suppress cell growth; and (f ) faster induction of apoptosis. Thus, CTS1 is an improved tumor suppressor and an alternative for the treatment of wild-type p53-resistant human tumors by gene therapy.

No abstract

To understand and dissect the mechanisms driving NK cell proliferation, we took advantage of the methodology used in cell therapy to numerically expand NK cells in the presence of K562-derived artificial Antigen Presenting Cells (aAPCs) and cytokines. For 9 consecutive weeks, high expression of CD137L by a K562-derived aAPC cell line was able to sustain NK cell expansion by 100 million-fold whereas low expression of CD137L by the parental K562 cell line supported the expansion by only 40,000-fold. The level of expression of CD137L, however, did not modulate the sensitivity of the K562 cell line to the intrinsic cytotoxicity of NK cells. Similarly, the low NK cell proliferation in the presence of the parental K562 cell line and cytokines was increased by adding agonistic anti-CD137 antibodies to levels similar to CD137L-expressing K562-derived aAPCs. Finally, the synergy between IL-15 and IL-21 was observed only upon CD137 engagement and the presence of aAPCs. Therefore, we conclude that NK cell proliferation requires cell-to-cell contact, the activation of CD137 axis, the presence of IL-15 (or its membranous form) and IL-21. By analogy with the three signals model required to activate T cells, we speculate, that the cell-to-cell contact represents “signal 1”, CD137 would be “signal 2” and cytokines would be “signal 3”. The precise nature of signal 1 remains to be defined. Citation Format: Laurent Vidard, Christine Dureuil-Sizaire, Jérémy Baudhuin, Lionel Vescovi, Laurence Durand, Véronique Sierra, Eric Parmantier. CD137 (4-1BB) engagement fine-tunes the synergistic IL-15- and IL-21-driven NK cell proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1524.

The LIN28A and LIN28B oncogenes are overexpressed in about 15% of human cancers, and they selectively block the expression of the tumor suppressor miRNA let-7 family, comprising twelve members (let-7a-1, -2, -3, let-7b, let-7c, let-7d, let-7e, let-7f-1, -2, let-7g, let-7i and miR-98) expressed from eight distinct loci. Upon binding to pre-let-7, LIN28A recruits ZCCHC11, a 3′ terminal uridylyl transferase, responsible for let-7 poly-uridylation and subsequent targeting of poly-uridylated let-7 miRNA for degradation. Expression of several let-7 miRNA family members was measured using quantitative RT-PCR in LIN28-positive or -negative tumor cell lines. In agreement with the inverse relationship between LIN28A/B and let-7 expression already observed by others (1), the lowest expression of let-7 miRNA was observed in LIN28A (e.g., IGROV-1 and T-47D) or LIN28B (e.g., NCI-H838, Hep-G2 and NCI-H1299) positive cell lines, and the degree of let-7 miRNA repression in LIN28A or LIN28B positive cell lines was particularly prominent for miR-98, let-7i and let-7b family members. Although the proliferation of LIN28B-positive tumor cell lines was sensitive to the 3 LIN28B siRNAs tested, the expression of LIN28A was not required for the proliferation and survival of LIN28A-expressing tumor cell lines, as demonstrated with one LIN28A siRNA that displayed an efficient knock-down at low concentrations with minimal impact on the proliferation of the T-47D tumor cell line. This LIN28A siRNA was further demonstrated to significantly upregulate the expression of several let-7 miRNAs such as miR-98 or let-7i, supporting an on-target modulation of the pathway. We also tested several siRNAs against ZCCHC11 uridylyl transferase, and found that the growth of either LIN28A- or LIN28B-positive tumor cell lines was inhibited by ZCCHC11 knockdown. However, ZCCHC11 protein knock-down was not able to restore let-7 miRNA expression, as it was seen for the LIN28A knock-down. These findings suggest that the role of ZCCHC11 in tumor cell lines might be more complex than just targeting poly-uridylated let-7 miRNA for degradation. Overall, our results seems to indicate that in LIN28A-positive tumor cell lines, LIN28A knock-down impacts let-7 miRNA expression, but has not a significant antiproliferative effect, whereas ZCCHC11 knock-down inhibits cell proliferation and this effect seems to be disconnected from let-7 miRNAs modulation. This lack of apparent effect on the expression of the miRNA let-7 expression might be related to the dual role of uridylyl transferases recently described for group II let-7 miRNAs, the mono- and poly-uridylation that leads to let-7 biogenesis and let-7 degradation respectively (2). 1- Viswanathan SR, Powers JT, Einhorn W, et al. Nat Genet 2009; 41: 843-848. 2- Heo I, Ha M, Lim J et al. Cell 2012; 151: 521-532. Citation Format: Laurent VIDARD, Claire MARIET, Eric BOITIER, Véronique SIERRA, Elisabeth CAVROIS, Carlos GARCIA-ECHEVERRIA, Hélène GOULAOUIC. Inhibition of either LIN28A or ZCCHC11 (TUT4) provides distinct effects on the expression of the let-7 miRNA family and tumor cell proliferation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3550. doi:10.1158/1538-7445.AM2014-3550