Mutually Exclusive Cdk4-Cyclin D1 and Cdk6-Cyclin D2 Pairing Inactivates Rb and Promotes Cell Cycle Dysregulation in Multiple Myeloma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 501-501
Author(s):  
Scott Ely ◽  
Maurizio Di Liberto ◽  
Ruben Niesvizky ◽  
Eunice Hatada ◽  
Hearn Cho ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cyclin D1 ◽  
Transcriptional Activation ◽  
Clinical Course ◽  
Cdk Inhibitors ◽  
Cell Cycle Regulators ◽  
Cyclin D2 ◽  
Constant Frequency

Abstract Multiple myeloma (MM), the second most common hematopoietic cancer, ultimately becomes refractory to treatment when self-renewing MM cells begin unrestrained proliferation by unknown mechanisms. Cell cycle reentry and progression is modulated by the balance between positive cell cycle regulators [cyclins and cyclin-dependent kinases (Cdk)] on the one hand and Cdk inhibitors (CKI) on the other. Here we show that one, but not more than one, of the three early G1 D cyclins is elevated in each case of MM. However, cyclin D1 or D3 overexpression rarely leads to phosphorylation of the retinoblastoma protein Rb and G1 cell cycle progression, unless cyclin-dependent kinase 4 (Cdk4) is correspondingly elevated in the absence of Cdk6. By contrast, cyclin D2 and Cdk6 are coordinately increased, thereby overriding inhibition by Cdk inhibitors, p18INK4c and p27Kip1, and phosphorylating Rb in conjunction with Cdk4. Thus, Cdk4 pairs exclusively with cyclin D1 whereas Cdk6 pairs exclusively with cyclin D2, although Cdk4 can also pair with cyclin D2 in MM cells. The basis for the exclusive Cdk4-cyclin D1 and Cdk6-cyclin D2 pairing is coordinated transcriptional activation. Cyclin D1 or D3 expression is scattered or uniform among bone marrow MM cells and at a constant frequency in the clinical course. By contrast, phosphorylation of Rb by Cdk6-cyclin D2 occurs in discrete foci of bone marrow MM cells early in the clinical course before cell proliferation, then is heightened with proliferation and disease progression. Mutually exclusive Cdk4-cyclin D1 and Cdk4/6-cyclin D2 pairing, therefore, critically controls cell cycle re-entry and G1 progression, which underlies the expansion of self-renewing MM cells in MM progression.

Proliferative Effect of APRIL On Primary Multiple Myeloma Cells Is Dictated by D-Type Cyclin Group and Translocation Status.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 124-124
Author(s):  
John Quinn ◽  
Janet Glassford ◽  
Laura Percy ◽  
Philippa Munson ◽  
Manuel Rodriguez-Justo ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cyclin D1 ◽  
Western Blotting ◽  
Proliferative Response ◽  
Cell Cycle Regulators ◽  
Cyclin D2 ◽  
Proliferative Effect ◽  
Membrane Bound

Abstract Abstract 124 Introduction: Understanding how multiple myeloma (MM) cells proliferate and self-renew is crucial to treating resistant disease and preventing relapse. The TNF family members APRIL (A proliferation-inducing ligand) and BAFF (B-cell activating factor) are secreted in the MM bone marrow microenvironment and share two common receptors, TACI and BCMA. APRIL and BAFF and have been shown to exert a mainly anti-apoptotic effect in human myeloma cell lines (HMCLs). Little is known, however, about the proliferative effect of APRIL and BAFF on primary MM cells. Aims: We aimed to determine the effect of APRIL and BAFF on cell cycle progression in primary MM cells and on the modulation of D-type cyclins and other cell cycle proteins. Patients and Methods: We studied the effects of APRIL and BAFF on purified MM cells from 26 patients by flow-cytometry using surface CD138 / Ki67 / propidium iodide staining. A culture system was optimised in which a mean (±SEM) of 73.3±10.9% (n=23) of primary CD138+ MM cells survived for up to 3 days in vitro. D-type cyclin expression was assessed by immuno-histochemistry (IHC) ± western blotting and correlated with FISH for IgH translocations. Fourteen patients expressed cyclin D1, 4 in association with t(11;14); while 12 patients expressed cyclin D2, 1 with t(4;14) and 4 with t(14;16). TACI and BCMA expression were determined by flow cytometry. Western blotting was used to determine expression and modulation of cell cycle proteins and activation of signalling pathways in vitro. Membrane-bound APRIL was detected by IHC in MM trephine biopsies. Results: In-vitro culture with APRIL for 72 hours increased the overall percentage of CD138+ cells in S+G2/M phases (S/G2M) from a mean (±SEM) of 5.5 ± 1.1% to 8.1 ± 1.5% (p<0.05). However, cyclin D2 expressing CD138+ cells demonstrated a greater response to APRIL than D1 expressing cells. In the D2 group (n=12), APRIL increased the S/G2M fraction from 8.3 ± 2.1% to 13.5 ± 2.2%, (p<0.01), while in the D1 group (n=14), proliferation was unaffected (3.0 ± 0.5%, compared with 3.1 ± 0.7% in control). Similar results were obtained when absolute numbers of CD138+ cells in S/G2M were analysed. Proliferative response to APRIL was more marked in those cases with IgH translocations (S/G2M fraction increased from 6.4±1.6% to 14.8±2.0%, p<0.001). In comparison with APRIL, BAFF had a lesser effect, increasing the S/G2M fraction in the D2 group from 8.3 ± 2.1% in control to 11.7 ± 3.5%, p=0.05). BAFF had no significant effect on proliferation in the D1 group. Results were verified by increased 3H-thymidine and bromodeoxyuridine uptake. APRIL-induced proliferation was maximal after 48-72 hours of incubation, was dose-dependent (maximal effect at 200ng/ml) and completely inhibited by TACI-Fc. In the D2 group, proliferation in response to APRIL was accompanied by increased expression of cyclin D2, cdk4, cdk6 and phospho-pRB by western blotting. In contrast, in the D1 group, neither cyclin D1 nor other cell cycle regulators was affected by APRIL. Proliferative response to APRIL was accompanied by increased expression of phospo-PKB. APRIL-induced proliferation showed a positive correlation with TACI expression (p<0.01), but not with BCMA. In contrast to the cell cycle effect, APRIL had no significant effect on survival of primary CD138+ cells either in D2 or D1 groups (viable cell number was 91±22%, and 129±18% of control respectively). IHC revealed the presence of membrane-bound APRIL on MM cells in BM sections from both D1 and D2 patients. IHC was used to examine expression of cell cycle regulators in vivo. In 5 cases where cyclin D1 expression was associated with proliferation, ie phospho-pRb expression, strong expression of cdk6 was seen, with weaker expression of cdk4. Expression of cyclin D2 was always accompanied by phospho-pRb expression (n=5), with strong expression of both cdk4 and -6 in 3, and just cdk6 in 2. Conclusions: APRIL stimulates the proliferation of cyclin D2 expressing primary MM cells, in particular those associated with known IgH translocations, but has minimal effect on cells expressing cyclin D1. These findings suggest that MM cells from different cyclin D/translocation classes rely on different stimuli for proliferation and cell-cycle re-entry. Disclosures: No relevant conflicts of interest to declare.

APRIL promotes cell-cycle progression in primary multiple myeloma cells: influence of D-type cyclin group and translocation status

Blood ◽  
2011 ◽  
Vol 117 (3) ◽  
pp. 890-901 ◽  
Author(s):  
John Quinn ◽  
Janet Glassford ◽  
Laura Percy ◽  
Philippa Munson ◽  
Teresa Marafioti ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cyclin D1 ◽  
Cell Cycle Progression ◽  
Surface Expression ◽  
Nuclear Factor Κb ◽  
Cyclin D2 ◽  
B Cell Maturation ◽  
M Phase ◽  
Protein Cell

Abstract A proliferation-inducing ligand (APRIL) promotes survival and drug resistance in multiple myeloma (MM) cell lines. We studied the effect of APRIL on cell-cycle behavior in primary MM cells and correlated our findings with D-type cyclin expression by immunohistochemistry and/or Western blotting. In MM cases, expressing cyclin D2 APRIL significantly increased the percentage of CD138+ cells in S + G2/M phase (from 8.4% ± 1.9% to 14.3% ± 2.6%, n = 15, P < .01), whereas a lesser effect was seen in cases expressing cyclin D1 (n = 18). Cell-cycle response to APRIL was most marked for cyclin D2-expressing cases with IgH translocations (P < .01) and was accompanied by increased expression of cyclin D2, CDK4, CDK6, and phospho-retinoblastoma protein. Cell-cycle proteins in cyclin D1+ cells were not modulated by APRIL. Surface expression of B-cell maturation antigen and transmembrane activator and calcium-modulating cyclophilin ligand interactor was not significantly different between cyclin D1+ and D2+ MM cells. We observed activation of nuclear factor-κB and PI3-kinase pathways in response to APRIL in both cyclin D1+ and D2+ MM cells. In conclusion, APRIL stimulates G1/S progression in cyclin D2+ MM cells bearing IgH translocations but has minimal effect on cyclin D1+ cells, suggesting MM cells from different cyclin D/translocation classes rely on different mechanisms for cell-cycle re-entry.

Targeting Cyclin D1 in the Treatment of Multiple Myeloma: Preclinical Validation of a Novel Specific Small Molecule Cyclin D1 Inhibitor, P276-00.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3454-3454
Author(s):  
Noopur Raje ◽  
Teru Hideshima ◽  
Sonia Vallet ◽  
Shweta Chhetri ◽  
Constantine Mitsiades ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Phase I ◽  
Cyclin D1 ◽  
Therapeutic Target ◽  
Growth Arrest

Abstract Either an overexpression or dysregulation of cyclin D1, D2, or D3, has been reported in the majority of multiple myeloma (MM) tumors, suggesting a possible early unifying event in MM pathogenesis. This proposed critical role of cyclin D dysregulation in myeloma pathogenesis makes the cyclins, specifically cyclin D1, an attractive therapeutic target. We have evaluated a specific small molecule cyclin D1 inhibitor, P276-00 in MM. Its specificity has been confirmed in an in vitro kinase assay by potent inhibitory activity for Cdk4-D1 as compared to Cdk2-E. Additionally in vitro kinase assays against a broad range of other kinases have also confirmed specificity for D1 and B cyclins at nanomolar concentrations. P276-00 has been tested against a wide range of cancer cell types in both in vitro and tumor xenograft models. Based on these data, it is undergoing phase I clinical testing in North America. We have observed both time and dose dependent in vitro activity against a broad range of MM cells sensitive and resistant to conventional agents like dexamethasone, doxorubicin, and melphalan with IC50 ranging from 400–800nM. Spectral karyotyping confirmed t(11;14) (q13;q32) in KMS 12 MM cells which were sensitive to P276-00. Importantly, it has demonstrated activity in primary patient derived tumor cells. Cell cycle analysis confirmed that P276-00 induced either growth arrest or apoptosis in MM cells depending on the cell line. Apoptosis was in part caspase dependent suggested by partial reversal of cytotoxicity by Z-VAD Fmk. P276-00 inhibited Rb-1 phosphorylation as early as 6 hours in most of the MM cell lines tested associated with a decrease in cdk4 suggesting a regulatory role of P276-00 in cell cycle progression. These changes preceeded growth arrest and apoptosis of MM cells on cell cycle analysis. Ongoing studies are using SiRNA to Cyclin D1 to confirm this regulatory role of P276-00. As cyclin D1 dysregulation or overexpression can render MM cells more susceptible to proliferative stimuli such as IL-6, IGF-1, and the bone marrow microenvironment, we tested the effects of P276-00 in the presence of these cytokines and bone marrow stromal cells (BMSCs). Our data confirms that P276-00 was able to overcome these proliferative signals and induce apoptosis in MM cells. Next we evaluated in vivo efficacy of P276-00 in NOD-SCID mice bearing GFP+ MM xenografts. Animals were treated with either control PBS or P276-00 intraperitoneally at 25 mg/kg three times a week for 3 weeks. Our data confirms in vivo anti-tumor activity of P276-00 as suggested by a significant decrease in biluminesence of GFP+ MM cells (p<0.05) and a decrease in tumor volume. Immunohistochemistry on tumor tissue from P76.00 treated, and control animals validates our in vitro studies and will be presented. In vitro combination studies with bortezomib have been completed suggesting synergism. P276-00 and bortezomib combination is currently being tested in our in vivo model. These studies confirm cyclin D1 to be an important therapeutic target in MM and form the basis of a phase I/II study of P276-00 alone and in combination in the treatment of MM.

Proteomic Analysis of Multiple Myeloma Identifies Potential Targets for Drug Therapy.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2446-2446
Author(s):  
Irene M. Ghobrial ◽  
Jeanette E. Eckel ◽  
Alexey A. Leontovich ◽  
Michael Timm ◽  
Gregory Ahmann ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Protein Expression ◽  
Signaling Pathway ◽  
Plasma Cells ◽  
Differentially Expressed ◽  
Ras Signaling ◽  
Cell Cycle Regulators ◽  
Group 3

Abstract The objective of this study was to investigate the underlying molecular alteration in multiple myeloma at the protein level in order to identify regulators of pathogenesis, discover novel targets of therapy, and compare genetic and proteomic alterations. We employed antibody protein microarrays (BD Clonetech, CA) to measure changes in the patterns of protein expression between MM and normal plasma cells. The antibody array is a new technique enabling protein differences to be assayed directly by hybridizing fluorescently labeled protein mixtures from cell extracts onto glass slides spotted with 512 different monoclonal antibodies. CD138+purified plasma cells were obtained from cryopreserved bone marrow samples of 12 newly diagnosed patients with MM. The labeling index was high (1% cutoff) in 7 samples and low in the other 5. Control plasma cells were obtained from 9 pooled CD138+ purified normal donor bone marrow plasma cells. Interphase FISH analysis for 17p deletion, 13q deletion, and t(11:14) were performed. To assess differential expression, the mean of the ratios of Cy5/Cy3 for each sample were analyzed using the Clontech software to calculate an internally normalized ratio. The normalized data were analyzed by the Genespring software. Unsupervised clustering identified 4 groups of MM. Changes of protein expression ≥2 fold in 70% of the samples as compared to control were identified. There were 6 proteins differentially expressed between all MM samples and control cells including proteins in the ras signaling pathway (KSR-1), the ubiquitin pathway (Ubc-H6), cyclin-dependent kinases (CDK4), cytokines (IL-6), DNA toposisomerase II, and the rho-interacting serine-threonine kinase CRIK. Proteins differentially expressed in MM groups 1 and 2 compared to normal control included cell cycle regulators (cul-2, MCM6, PCNA, TGFb1), kinases (p70S6K, PKC), and chromatin regulators (Ran, AKAP450, Rad50). Protiens differentially expressed in MM group 3 included cell cycle regulators (CDK2, CLK1, MENA), apoptosis regulators (XIAP, caspase 4, perforin) kinases (IKKa and RAC1 in the Wnt signaling pathway) and P53 regulators, while proteins identified in MM group 4 included NFkB/ubiquitin proteins (IKKa and Ubch6), cell cycle regulators (c-myc, CDK4), p53 pathway proteins (53bp2), ras-signaling proteins (KSR1), and the kinase CRIK. There were no differences in protein expression between the high and low labeling index groups. 13q was identified in 5 (42%), 17 p in 1(8%) and t(11:14) in 1(8%) patients. 80% of the 13 q deletion cases clustered in MM group 1 and 2 patients. Cyclin D-1 was upregulated in 5 (42%) patients including the patient with (11:14) translocation. This is the first proteomic study of patients with MM. The results are consistent with previously identified genetic alterations in MM indicating that this novel technique could be used in identifying molecular changes in MM. It identifies novel proteins dysregulated in MM that differ between the 4 MM groups. These results may be used in the future to individualize therapy based on the proteins dysregulated in each group. For example, IKK inhibitors may be useful in group 3 MM patients, while mTOR inhibitors (upstream of p70S6K) could be used in groups 1 and 2 patients. Future correlations with gene expression arrays and prognosis in a larger cohort of patients is warranted.

scholarly journals Oct4/Sox2-Regulated miR-302 Targets Cyclin D1 in Human Embryonic Stem Cells

2008 ◽  
Vol 28 (20) ◽  
pp. 6426-6438 ◽  
Author(s):  
Deborah A. Greer Card ◽  
Pratibha B. Hebbar ◽  
Leping Li ◽  
Kevin W. Trotter ◽  
Yoshihiro Komatsu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cyclin D1 ◽  
Transcriptional Activation ◽  
Developmental Stages ◽  
Embryonic Stem ◽  
Cell Cycle Regulators ◽  
Human Escs ◽  
Pluripotent Cells ◽  
Transformed Cell Lines ◽  
Functional Mechanisms

ABSTRACT Oct4 and Sox2 are transcription factors required for pluripotency during early embryogenesis and for the maintenance of embryonic stem cell (ESC) identity. Functional mechanisms contributing to pluripotency are expected to be associated with genes transcriptionally activated by these factors. Here, we show that Oct4 and Sox2 bind to a conserved promoter region of miR-302, a cluster of eight microRNAs expressed specifically in ESCs and pluripotent cells. The expression of miR-302a is dependent on Oct4/Sox2 in human ESCs (hESCs), and miR-302a is expressed at the same developmental stages and in the same tissues as Oct4 during embryogenesis. miR-302a is predicted to target many cell cycle regulators, and the expression of miR-302a in primary and transformed cell lines promotes an increase in S-phase and a decrease in G1-phase cells, reminiscent of an ESC-like cell cycle profile. Correspondingly, the inhibition of miR-302 causes hESCs to accumulate in G1 phase. Moreover, we show that miR-302a represses the productive translation of an important G1 regulator, cyclin D1, in hESCs. The transcriptional activation of miR-302 and the translational repression of its targets, such as cyclin D1, may provide a link between Oct4/Sox2 and cell cycle regulation in pluripotent cells.

scholarly journals Increased expression of cyclin-D1 on trephine bone marrow biopsies independently predicts for shorter overall survival in patients with multiple myeloma treated with novel agents

2012 ◽  
Vol 87 (7) ◽  
pp. 734-736 ◽  
Author(s):  
Anna Tasidou ◽  
Maria Roussou ◽  
Evangelos Terpos ◽  
Efstathios Kastritis ◽  
Maria Gkotzamanidou ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Overall Survival ◽  
Cyclin D1 ◽  
Novel Agents ◽  
Bone Marrow Biopsies

scholarly journals The Necessary for Long-Term Follow-up of Mutated Genes and Clonal Evolutions in Multiple Myeloma Combined with cfDNA Assay

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5515-5515
Author(s):  
Yuko Mishima ◽  
Yuji Mishima ◽  
Masahiro Yokoyama ◽  
Noriko Nishimura ◽  
Yoshiharu Kusano ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Genomic Dna ◽  
Research Funding ◽  
Somatic Mutations ◽  
Clinical Course ◽  
Bone Marrow Aspiration ◽  
Long Term Follow Up

Introduction)Somatic mutations in multiple myeloma (MM) are strongly related to the clinical outcome and clonal evolution over the clinical course, and are a major problem. From a clinical viewpoint, although numerous novel drugs have been utilized, achieving long-lasting and complete remission remains difficult. Recent studies have elucidated the mutated genes using next-generation sequencing, and have examined how clonal change can be acquired in myeloma. In this study, we traced the transition of the somatic mutations of bone marrow tumor cells in patients with MM over a long-term follow-up. Furthermore, we compared the somatic mutations found in serum cell-free DNA (cfDNA) and mutated genes obtained from bone marrow myeloma cells. Material and Methods)Patients diagnosed with multiple myeloma who provided written informed consent to participate in the study were enrolled. Patients were treated by immuno-chemotherapy with or without radiation between 2000 and 2017 at our institute. Bone marrow aspiration and biopsy were performed at the time of diagnosis and upon disease progression. Around the time of bone marrow aspiration, serum was obtained from a peripheral blood sample for cfDNA analysis. Myeloma cells were separated from bone marrow samples with MicroBeads of CD138 antibody and genomic DNA was extracted. The peripheral blood samples derived from myeloma patients. The cfDNA was extracted from the serum using a Maxwell RSC cfDNA Plasma kit. Using genomic DNA derived from cfDNA and bone marrow, multiplex polymerase chain reaction (PCR) was performed, and a sequence library was then constructed with an Ion Custom Amplicon panel. The panel for the sequence library was designed using an Ion AmpliSeq DesignerTM. 126 targeted genes were selected. The genomes were sequenced using the Ion ProtonTM System. This protocol was approved by the institutional review board and the Genomic Review Board of the Japanese Foundation for Cancer Research. Result)We followed 7 patients' long term-clinical course and the transition of mutations (8.5 year average). The expression of myeloma driver genes, such as RAS, BRAF, and MYC, were not critical. We did, however, detect a relationship between an increase in the dominant mutated gene, such as TP53, DIS3, FAM46C, KDM6B, and EGR1 and poor prognosis in patients with myeloma. Next, we calculated the cfDNA concentrations from 34 cases. The cfDNA concentrations were significantly higher than 10 control cases (average 62.0 ng/mL (0-200 ng/mL) and 8.18 ng/mL (4.3-14.1 ng/mL), P=0.0046). The 2.5 year-progression free survival (PFS) during the first treatment of MM were tend to be poorer in the group with cfDNA>50 ng/mL (72.9%) than the group with cfDNA<50 ng/mL(25.9%), however there are no statistical significance (P = 0.15).We caluculated concordance rate of derived mutations from bone marrow MM cells and cfDNA in 7 cases. The somatic mutations found in serum cell-free DNA (cfDNA) and bone marrow MM cells were determined the correlation coefficients. However, there are few difference expression pattern in each source. In cfDNA assay, CREEP, EGR1, HDAC4, HDAC6, and JMJD1C were highly expressed as 57.1% (4/7) - 85.7% (6/7), and these results were almost the same as those for bone marrow MM cells. On the other hand, KDM1A (85.7%), PI3KCD (71.4%), and KDM3B (57.1%) were highly detected in cfDNA, although those were not frequently expressed in bone marrow. Discussion)Our data demonstrate the importance of the long-term follow-up of somatic mutations during the clinical course of myeloma. Serum cfDNA is a useful alternative source for detecting somatic mutations in MM patients during long-term follow-up. Disclosures Mishima: Chugai-Roche Pharmaceuticals Co.,Ltd.: Consultancy. Yokoyama:Chugai-Roche Pharmaceuticals Co.,Ltd.: Consultancy. Nishimura:Chugai-Roche Pharmaceuticals Co.,Ltd.: Consultancy; Celgene K.K.: Honoraria. Hatake:Celgene K.K.: Research Funding; Janssen Pharmaceutical K.K.: Research Funding; Takeda Pharmaceutical Co.,Ltd.: Honoraria. Terui:Bristol-Myers Squibb K.K.: Research Funding; Bristol-Myers Squibb, Celgene, Janssen, Takeda, MSD, Eisai, Ono, and Chugai-Roche Pharmaceuticals Co.,Ltd.: Honoraria.

scholarly journals Immunohistochemical detection of cell cycle regulators, Fhit protein and apoptotic cells in parathyroid lesions

2003 ◽  
pp. 81-87 ◽  
Author(s):  
GE Thomopoulou ◽  
S Tseleni-Balafouta ◽  
AC Lazaris ◽  
H Koutselini ◽  
N Kavantzas ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cyclin D1 ◽  
Cell Cycle Control ◽  
Parathyroid Glands ◽  
Considerable Proportion ◽  
Parathyroid Cell ◽  
Apoptotic Cells ◽  
Cell Cycle Regulators ◽  
Suppressor Activity ◽  
Fhit Protein

OBJECTIVE: The pathological distinction between parathyroid neoplasms and hyperplasias remains difficult. Changes in cell cycle control may lead to clonal proliferation and precede tumorigenesis. The parathyroid adenoma 1 oncogene, subsequently identified as the gene encoding cyclin D1, has been shown to be important to parathyroid tumour development. In addition to cell proliferation, the mechanisms of parathyroid cell turnover include apoptosis. The tumour-suppressor activity of the fragile histidine triad gene (FHIT) is linked to its proapoptotic function and cell cycle control. We attempted to evaluate the cellular proliferative kinetics and apoptotic function of the parathyroid glands in patients with non-familial hyperparathyroidism (HPT). DESIGN: TIssue specimens were taken from 40 patients with primary HPT (17 adenomas, two carcinomas and 21 primary hyperplasias) and from 30 patients with secondary HPT. Normal glands served as controls. METHODS: In a standard immunohistochemical procedure, monoclonal antibodies to Ki-67 antigen and single-stranded DNA were applied to detect cycling and apoptotic cells respectively; polyclonal antibodies to cyclin D1 and Fhit protein were used. Immunostaining was estimated by image analysis and statistical analysis was subsequently performed. RESULTS: Significantly higher proliferative and apoptotic indexes were detected in the diseased glands in comparison with normal controls. In neoplastic and secondarily hyperplastic glands, apoptotic indexes were higher than in primarily hyperplastic glands; the difference between neoplastic and primarily hyperplastic glands was statistically significant (P=0.034). Cyclin D1 was overexpressed in a considerable proportion of tumours (68.4%). A reduction of Fhit protein immunoreactivity was selectively noticed in carcinomas. CONCLUSIONS: In primary hyperplasia, the remarkable proliferation of parathyroid glands may be due to the reduction of the apoptotic process. FHIT gene abnormalities are worthy of investigation in parathyroid carcinogenesis.

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