Expression of Hpttg Proto-Oncogene in Lymphoid Neoplasias

Expression of hpttg proto-oncogene in lymphoid neoplasias

Carmen Sa´ ez1, Teresa Pereda1, Juan J Borrero1, Agueda Espina2, Francisco Romero3, Marı´ a Tortolero3, Jose´ A Pintor-Toro2, Dolores I Segura1 and Miguel A Japo´ n*,1

1Department of Pathology, Hospital Universitario Virgen del Rocı´o, Seville, 41013 Spain; 2Instituto de Recursos Naturales y Agrobiologı´a, CSIC, Seville, 41080 Spain; 3Department of Microbiology, Faculty of Biology, University of Seville, Seville, 41080 Spain

Pituitary tumor-transforming gene (pttg) is a distinct (Pei and Melmed, 1997). A human homolog of pttg, proto-oncogene which is expressed in certain normal hpttg, was first cloned from a thymus cDNA library tissues with high proliferation rate and in a variety of (Domı´ nguez et al., 1998). hpttg gene is expressed in tumors. PTTG is the vertebrate analog of yeast securins pituitary adenomas as well as in other epithelial tumors Pds1 and Cut2 with a key role in the regulation of sister such as breast, lung, and colorectal carcinomas (Sa´ ez et chromatid separation during mitosis. Impairment of al., 1999; Zhang et al., 1999a; Heaney et al., 2000). We PTTG regulated functions is expected to lead to have demonstrated a correlation between hpttg expres- chromosomal instability and aneuploidy. Human pttg sion and cell proliferation in vitro. hPTTG protein level (hpttg) is abundantly expressed in Jurkat T lympho- is up-regulated in rapidly proliferating cells, is down- blastic lymphoma cells but not in normal peripheral regulated in response to serum starvation or cell blood leukocytes. To obtain additional data on the confluence, and is regulated in a cell cycle-dependent potential role of hpttg in lymphomagenesis we selected manner peaking in mitosis (Ramos-Morales et al., 150 cases of lymphoid tumors for the assessment of hpttg 2000). hpttg has been mapped to chromosome 5q33 expression in tumor tissues. Immunohistochemical studies (Zhang et al., 1999b), a region that is associated with on formalin-fixed, paraffin-embedded tissues revealed reports of neoplastic diseases, including myeloid hPTTG in 38.8% of B-cell lymphomas, 70.2% of T- leukemia, chronic myeloproliferative disorders, and cell lymphomas, and 73.1% of Hodgkin’s lymphomas. myelodisplastic syndromes (Strausberg et al., 1997). Among B-cell lymphomas, the most frequently immu- hPTTG is a protein of 202 amino acids that has a nostained tumors were plasma cell tumors, diffuse large basic N-terminal portion and an acidic C-terminal cell lymphomas, and follicle center cell lymphomas. In portion (Domı´ nguez et al., 1998). It has several Hodgkin’s disease, immunoreactivity was mainly noted in putative SH3-binding sites (Domı´ nguez et al., 1998) Reed-Sternberg cells. In conclusion, the frequent over- and a conserved motif localized in the N-terminal expression of hpttg in many histological subtypes of portion matches the destruction box (D-box) shared by lymphoma suggests the involvement of this proto- many anaphase-promoting complex or cyclosome oncogene in lymphomagenesis. substrates (King et al., 1996). Recently, hpttg gene Oncogene (2002) 21, 8173 – 8177. doi:10.1038/sj.onc. product has been identified as a vertebrate securin, 1205954 involved in the regulation of sister-chromatid separation during mitosis (Zou et al., 1999). Defective Keywords: hpttg proto-oncogene; B-cell lymphoma; T- separation could lead to chromosome missegregation cell lymphoma; Hodgkin’s disease; immunohistochem- and aneuploidy. On this basis, genomic imbalance as a istry result of chromosome missegregation provides an explanation for the oncogenic potential of dysregulated hpttg expression (Jallepalli et al., 2001). Gain of Pituitary tumor-transforming gene (pttg) was originally oncogene dosage and loss of tumor supressor genes isolated from clonal rat GH4 pituitary tumor cells. are increasingly being identified as frequent events in This gene is diferentially expressed in tumorous but not the lymphomatous process (Werner et al., 1997; Rao et in normal pituitary cells, and its overexpression in al., 1998). The fact that Jurkat lymphoblastic T cells NIH3T3 mouse fibroblasts is able to induce cell express high levels of hpttg mRNA, in contrast to its transformation in vitro and tumor formation in vivo undetectable expression in peripheral blood lympho- when transformed cells are injected into athymic mice cytes (Domı´ nguez et al., 1998), prompted us to assess the expression of hpttg in tissues affected by malignant lymphoproliferative disorders, both at the mRNA and protein levels. The availability of a policlonal anti- *Correspondence: MA Japo´ n, Department of Pathology, Hospital hPTTG antibody (Domı´ nguez et al., 1998) that Universitario Virgen del Rocı´ o, Avenida Manuel Siurot s/n, Seville, 41013 Spain; E-mail: [email protected] efficiently works in paraffin sections allowed us to Received 22 January 2002; revised 8 August 2002; accepted 8 determine the expression of hPTTG in a series of 150 August 2002 lymphomas by immunohistochemistry. The specificity hpttg expression in lymphomas CSaéz et al 8174 of this antibody has been previously demonstrated with and variants were markedly labeled by anti-hPTTG the use of immunoabsorption controls (Sa´ ez et al., antibody and became easily distinguished from the 1999). background of small lymphocytes that showed very All tumors were collected from the archives of the low levels of hPTTG expression (Figure 1k). Expres- Department of Pathology, reviewed by two indepen- sion of hpttg mRNA was also analysed in selected dent pathologists and classified according to the cases by in situ hybridization on cryostat tissue Revised European-American Lymphoma (REAL) clas- sections. Using a digoxigenin-labeled antisense hpttg sification (Harris et al., 1994). Table 1 summarizes the riboprobe we demonstrated an intense hpttg mRNA results of immunostaining. In this series, we found that signal in the cytoplasm of Reed-Sternberg cells (Figure most Hodgkin’s disease (73.1%) and non-Hodgkin’s T- 1l). Expression of hpttg mRNA was also confirmed in cell lymphoma (70.2%) tissues have remarkable levels available cryostat sections from B-cell lymphomas of of hPTTG. The frequency of detectable immunohisto- the follicular and diffuse large cell subtypes. In chemical expression of hPTTG was lower in non- addition, expression of hpttg was analysed by North- Hodgkin’s B-cell lymphoma tissues (38.8%). Among ern blot in 35 cases of the series with available frozen B-cell lymphomas (Figure 1a – f), plasma cell neopla- tissues. In Northern blots (Figure 2), detectable levels sias (seven out of 10), follicular lymphomas (eight out of hpttg mRNA were found in most cases studied (27 of 18), and diffuse large cell lymphomas (six out of 14) out of 35). Densitometric quantitation yielded variable were found to be the most frequent subtypes hpttg mRNA to 18S RNA ratios within each group of expressing detectable levels of hPTTG. Other B-cell lymphoma subtypes (e.g. 0.14 – 1.06, mean 0.66 for malignancies analysed had either very weak or absent follicular lymphomas; 0.73 – 1.25, mean 0.86 for diffuse immunoreactivity for hPTTG in most cases. Only one large B-cell lymphomas). Levels of hpttg mRNA out of six mantle cell lymphomas, two out of nine correlated with the intensity of immunostaining in Burkitt’s lymphomas, and two out of 10 MALT these cases, which suggests that upregulation of hpttg lymphomas had immunohistochemically detectable occurs at the transcriptional level. Expression of hpttg levels of hPTTG. None of the three small lymphocytic mRNA was not detected on blots in samples from lymphomas were positive for hPTTG. Overall, T cell normal lymph nodes, but was noted in one case of lymphomas were the most heavily immunostained with lymphoid hyperplasia. By immunohistochemistry anti-hPTTG antibody. Positivity was very intense in 10 hPTTG was observed in cells of the germinal centers cases: one nasal angiocentric lymphoma, five periph- in cases of lymphoid hyperplasia. Taken together, eral T-cell lymphomas, three mycosis fungoides, and these data suggest that hpttg is involved in the one case of anaplastic large cell lymphoma (Figure neoplastic process of many histological subtypes of 1g – j). In Hodgkin’s lymphomas, Reed-Sternberg cells lymphomas.

Table 1 Immunohistochemical expression of hPTTG in lymphoid neoplasms Immunohistochemistrya Per cent No. of cases 7 + ++ positivity

B-cell neoplasms Precursor lymphoblastic lymphoma 1 0 1 0 Small lymphocytic lymphoma 3 3 0 0 Lymphoplasmacytoid lymphoma 2 1 1 0 Mantle cell lymphoma 6 5 1 0 Follicular lymphoma 14 8 4 2 Marginal zone lymphoma (MALT type) 10 8 2 0 Plasmacytoma/Plasma cell myeloma 10 3 6 1 Diffuse large B-cell lymphoma 18 10 6 2 Burkitt’s lymphoma 8 6 2 0 Total 72 44 23 5 38.8 T-cell neoplasms Precursor lymphoblastic lymphoma 1 1 0 0 Mycosis fungoides 4 0 1 3 Peripheral T-cell lymphoma (unspecified) 21 6 10 5 Angioimmunoblastic lymphoma 3 1 2 0 Angiocentric lymphoma 3 1 1 1 Anaplastic large cell lymphoma 5 2 2 1 Total 37 11 16 10 70.2 Hodgkin’s disease Nodular sclerosis 27 5 15 7 Mixed cellularity 9 3 3 3 Lymphocyte depletion 2 1 1 0 Lymphocyte predominance 3 2 0 1 Total 41 11 19 11 73.1

aImmunostaining was scored as follows: 7, absence of staining or weak immunostaining in less than 10% of cells; +, weak to moderate immunostaining in more than 10% of cells; ++, intense immunostaining in more than 10% of cells

Oncogene hpttg expression in lymphomas CSa´ez et al 8175

Figure 1 Analysis of hpttg expression in lymphoma tissue sections. (a – k) Immunohistochemical staining of hPTTG in lymphoma tissues. Four-mm-thick parafﬁn tissue sections were incubated overnight at 48C with polyclonal anti-hPTTG antibody (Domı´ nguez et al., 1998). Secondary biotinylated antibody, streptavidin-biotin-peroxidase, and 3-3’ diaminobencidine, all from Dako A/S (Glostr- up, Denmark), were applied to visualize immunostaining. Sections were counterstained with hematoxylin. Sections where primary antibody was omitted were used as negative controls. (a) Follicular lymphoma; (b) MALT lymphoma; (c) mantle cell lymphoma; (d) diffuse large B cell lymphoma; (e) plasmacytoma; (f) nonneoplastic lymph node; (g) peripheral T cell lymphoma; (h) anaplastic large T cell lymphoma; (i) angiocentric T cell lymphoma; (j) angioimmunoblastic T cell lymphoma; (k) Hodgkin’s lymphoma. (l) In situ hybridization of hpttg mRNA in Hodgkin’s lymphoma. Antisense hpttg digoxigenin (DIG)-labeled riboprobe was obtained by in vitro transcription (Roche Molecular Biochemicals, Mannheim, Germany) using a plasmid containing 700 bp hpttg cDNA sequence as template. Five-mm-thick cryostat sections were ﬁxed in 4% paraformaldehyde in DEPC-treated PBS for 30 min. Hybridization and posthybridization conditions were those recommended by manufacturer’s protocols. Anti-DIG antibody coupled to alkaline phosphatase and NBT/BCIP were used for signal detection (Roche Molecular Biochemicals). Nuclear fast red was the counterstain. Sections hybridized under the same experimental conditions but with sense hpttg riboprobe were used as negative controls. Bars, 50 mm

Genetic instability in most cancers is observed at the segregation of chromosomes during mitosis can lead to chromosome level and results in losses and gains of chromosomal instability (Lengauer et al., 1998). In whole chromosomes leading to aneuplody. Alterations some cases this chromosomal instability is associated in genes involved in the processes of replication and with alterations in the mitotic spindle checkpoint

Oncogene hpttg expression in lymphomas CSaéz et al 8176 hPTTG with the Ku-70/Ku-80 heterodimer is prevented by the presence of DNA double-strand breaks indicating that the induction of DNA damage- response pathways might be connected with sister chromatid separation, in the way that inhibition of hPTTG-Ku interaction might favor the delay of the onset of mitosis while DNA repair takes place. Mice deficient in Ku-70 develop spontaneous thymic and disseminated T-cell lymphomas with multiple karyo- typic abnormalities (Li et al., 1998). It cannot be ruled out the possibility that both inactivation of ku-70 gene and defective regulation of PTTG leading to alterations in sister chromatid exchange and separation cooperate to facilitate lymphomagenesis. There are several lines of evidence that PTTG might also function in vivo as a transcriptional activator. The C-terminal region of hPTTG is able to transactivate when fused to a heterologous DNA-binding domain (Domı´ nguez et al., 1998). Also, cotransfection experi- Figure 2 Northern blot analysis of hpttg mRNA expression in ments with PTTG and PBF (PTTG binding factor) lymphoma tissue samples. Ten mg of total RNA from frozen tis- led to the transcriptional activation of the basic sue samples were electrophoresed in 1% agarose-formaldehyde gels, transferred onto nylon membranes, and hybridized to a fibroblast growth factor promoter (Chien and Pei, 32P-labeled full-length hpttg cDNA probe. Hybridizations to 18S 2000). Most interestingly, it has recently been reported probe are also shown as controls of RNA loading. Scanning den- that PTTG binds to c-myc promoter and activates c- sitometry was performed for quantitation using ScanAnalysis myc transcription in transfected cells (Pei, 2001). The software (Biosoft, Cambridge, UK). FCL, follicle center cell lymphoma; MCL, mantle cell lymphoma; MZL, marginal zone B-cell c-myc proto-oncogene product is a critical regulator of lymphoma; PCM, plasma cell myeloma; DLBL, diffuse large B- cell proliferation and its expression is frequently cell lymphoma; BL, Burkitt’s lymphoma; TCL, peripheral T-cell altered in human tumors (Marcu et al., 1992). lymphoma; HD, Hodgkin’ disease; Hyp, hyperplastic lymph node; Dysregulation of c-myc expression is a well known NLN, normal lymph node molecular event in most cases of Burkitt’s lymphoma, mouse plasmacytoma, as well as many large B cell (Cahill et al., 1998), which controls the proper bipolar lymphomas harboring translocation t(8;14) that juxta- segregation of duplicated sister chromatids before poses the c-myc gene and an immunoglobulin locus anaphase (Hardwick, 1998). PTTG regulated degrada- (Cory, 1986; Landanyi et al., 1991; Rabbits, 1994). In tion appears to be essential for the separation of sister Burkitt’s lymphoma, mutations may either abrogate chromatids and mitotic exit. Recently, it has been the negative regulation of c-myc transcription or demonstrated that PTTG is needed for maintaining increase the stability of the c-Myc protein leading to chromosomal stability in humans. The inactivation of increased c-Myc protein activity (Hecht and Aster, hpttg in a karyotypically stable colorectal cancer cell 2000). Also, c-Myc seems to play a role in the genesis line via homologous recombination led to retardation of T cell lymphomas since c-myc gene is disrupted and of chromosome separation and hpttg-deficient cells dysregulated in T cell tumors of v-myb infected exhibited high chromosome loss rates (Jallepalli et al., transgenic mice (Davies et al., 1999). The identifica- 2001). tion of PTTG as a transcriptional activator of c-myc DNA damage-induced cell cycle checkpoints are also may provide a new mechanism that contributes to the control mechanisms that prevent cells with DNA dysregulation of c-myc expression in lymphomas with damage from entering mitosis. Alterations in genes elevated levels of PTTG. involved in the pathways of these checkpoints have The identification of tumors that overexpress hpttg been also implicated in tumorigenesis (Dasika et al., could be of potential use in the clinical setting since 1999). In Saccharomyces cerevisiae, the DNA damage hpttg mRNA levels have been related to invasiveness in that elicits a mitotic checkpoint arrest induces the colorectal tumors (Heaney et al., 2000). Cell cycle- phosphorylation of Pds1 protein (Cohen-Fix and dependent expression of hpttg (Ramos-Morales et al., Koshland, 1997), the budding yeast homolog of 2000) could suggest a correlation between the state of hPTTG. We have recently reported that hPTTG proliferation and the expression of hpttg in tumors, but interacts with the DNA-dependent protein kinase certain slowly proliferating tumors (e.g. pituitary (DNA-PK) (Romero et al., 2001), a heterotrimeric adenomas) do express very high levels of hpttg.In enzyme complex involved in the repair of DNA our cases of follicular lymphomas and diffuse large B- double-strand breaks (Jin and Weaver, 1997). hPTTG cell lymphomas, that averaged low and high prolifera- specifically interacts with the Ku-70 subunit, and forms tion indices respectively, correlation between a complex with the Ku-70/Ku-80 heterodimer in intact proliferation and hPTTG expression was not cells. In addition, hPTTG is phosphorylated in vitro by evidenced. As a tumor marker, hPTTG could be of the DNA-PK catalytic subunit. This association of special interest in categorizing subgroups of lympho-

Oncogene hpttg expression in lymphomas CSa´ez et al 8177 mas with poor patient survival (peripheral T-cell Acknowledgments lymphomas, diﬀuse large B-cell lymphomas) and future This work has been supported by grants from Ministerio studies will help to determine the impact of hPTTG de Educacio´ n y Cultura, Spain (SAF99-0125-C03); Direc- overexpression in the prognosis and treatment of cio´ n General de Universidades e Investigacio´ n, Junta de lymphoma patients. Andalucı´ a; and Fundacio´ nAndex.

References

Cahill DP, Lengauer C, Yu J, Riggins GJ, Willson JK, Li GC, Ouyang H, Li X, Nagasawa H, Little JB, Chen DJ, Markowitz SD, Kinzler KW and Vogelstein B. (1998). Ling CC, Fuks Z and Cordon-Cardo C. (1998). Mol. Cell., Nature, 392, 300 – 303. 2, 1–8. Chien W and Pei L. (2000). J. Biol. Chem., 275, 19422 – Marcu KB, Bossone SA and Patel AJ. (1992). Annu. Rev. 19427. Biochem., 61, 809 – 860. Cohen-Fix O and Koshland D. (1997). Proc. Natl. Acad. Sci. Pei L. (2001). J. Biol. Chem., 276, 8484 – 8491. USA., 94, 14361 – 14366. Pei L and Melmed S. (1997). Mol. Endocrinol., 11, 433 – 441. Cory S. (1986). Adv. Cancer Res., 47, 189 – 234. Rabbits TH. (1994). Nature, 372, 143 – 149. Dasika GK, Lin SCJ, Zhao S, Sung P, Tomkinson A and Lee Ramos-Morales F, Domı´ nguez A, Romero F, Luna R, EYHP. (1999). Oncogene, 18, 7883 – 7899. Multon MC, Pintor-Toro JA and Tortolero M. (2000). Davies J, Badiani P and Weston K. (1999). Oncogene, 18, Oncogene, 19, 403 – 409. 3643 – 3647. Rao PH, Houldsworth J, Dyomina K, Parsa NZ, Cigudosa Domı´ nguez A, Ramos-Morales F, Romero F, Rios RM, JC, Louie DC, Popplewell L, Ofﬁt K, Jhanwar SC and Dreyfus F, Tortolero M and Pintor-Toro JA. (1998). Chaganti RS. (1998). Blood, 92, 234 – 240. Oncogene, 17, 2187 – 2193. Romero F, Multon MC, Ramos-Morales F, Domı´ nguez A, Hardwick KG. (1998). Trends Genet., 14, 1–4. Bernal JA, Pintor-Toro JA and Tortolero M. (2001). Harris NL, Jaffe ES, Stein H, Banks PM, Chan JKC, Cleary Nucleic Acids Res., 29, 1300 – 1307. ML,DelsolG,DeWolf-PeetersC,FaliniB,GatterKC, Sa´ ez C, Japo´ n MA, Ramos-Morales F, Romero F, Segura Grogan TM, Isaacson PG, Knowles DM, Mason DY, DI, Tortolero M and Pintor-Toro JA. (1999). Oncogene, Muller-Hermelink HK, Pileri SA, Piris MA, Ralfkiaer E 18, 5473 – 5476. and Warnke RA. (1994). Blood, 84, 1361 – 1392. Strausberg RL, Dahl CA and Klausner RD. (1997). Nat. Heaney AP, Singson R, McCabe CJ, Nelson V, Nakashima Genet., 15, 415 – 416. M and Melmed S. (2000). Lancet, 355, 716 – 719. Werner CA, Do¨ hner H, Joos S, Tru¨ mper LH, Baudis M, Hecht JL and Aster JC. (2000). J. Clin. Oncol., 18, 3707 – Barth TFE, Ott G, Mo¨ ller P, Lichter P and Bentz M. 3721. (1997). Am.J.Pathol.,151, 335 – 342. Jallepalli PV, Waizenegger IC, Bunz F, Langer S, Speicher Zhang X, Horwitz GA, Heaney AP, Nakashima M, Prezant MR, Peters JM, Kinzler KW, Vogelstein B and Lengauer TR, Bronstein MD and Melmed S. (1999a). J. Clin. C. (2001). Cell, 105, 445 – 457. Endocrinol. Metab., 84, 761 – 767. Jin S and Weaver DT. (1997). EMBO J., 16, 6874 – 6885. Zhang X, Horwitz GA, Prezant TR, Valentini A, Nakashima King RW, Glotzer M and Kirschner MW. (1996). Mol. Biol. M, Bronstein MD and Melmed S. (1999b). Mol. Cell., 7, 1343 – 1357. Endocrinol., 13, 156 – 166. Landanyi M, Ofﬁt K, Jhanwar SC, Filippa DA and Chaganti Zou H, McGarry TJ, Bernal T and Kirschner MW. (1999). RSK. (1991). Blood, 77, 1057 – 1063. Science, 285, 418 – 422. Lengauer C, Kinzler KW and Vogelstein B. (1998). Nature, 396, 643 – 649.

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