Limits...
Identification and characterization of retinoblastoma gene mutations disturbing apoptosis in human breast cancers.

Berge EO, Knappskog S, Geisler S, Staalesen V, Pacal M, Børresen-Dale AL, Puntervoll P, Lillehaug JR, Lønning PE - Mol. Cancer (2010)

Bottom Line: Multiple sequence alignment across species indicates the spacer to be evolutionary conserved.All three RB1 point mutations encoded nuclear proteins with impaired ability to induce apoptosis compared to wild-type pRb in vitro.Although rare, our findings suggest RB1 mutations to be of pathological importance potentially affecting sensitivity to mitomycin/anthracycline treatment in breast cancer.

View Article: PubMed Central - HTML - PubMed

Affiliation: Section of Oncology, Institute of Medicine, University of Bergen, Norway.

ABSTRACT

Background: The tumor suppressor pRb plays a key role regulating cell cycle arrest, and disturbances in the RB1 gene have been reported in different cancer forms. However, the literature reports contradictory findings with respect to a pro--versus anti--apoptotic role of pRb, and the consequence of alterations in RB1 to chemotherapy sensitivity remains unclear. This study is part of a project investigating alterations in pivotal genes as predictive factors to chemotherapy sensitivity in breast cancer.

Results: Analyzing 73 locally advanced (stage III) breast cancers, we identified two somatic and one germline single nucleotide changes, each leading to amino acid substitution in the pRb protein (Leu607Ile, Arg698Trp, and Arg621Cys, respectively). This is the first study reporting point mutations affecting RB1 in breast cancer tissue. In addition, MLPA analysis revealed two large multiexon deletions (exons 13 to 27 and exons 21 to 23) with the exons 21-23 deletion occurring in the tumor also harboring the Leu607Ile mutation. Interestingly, Leu607Ile and Arg621Cys point mutations both localize to the spacer region of the pRb protein, a region previously shown to harbor somatic and germline mutations. Multiple sequence alignment across species indicates the spacer to be evolutionary conserved. All three RB1 point mutations encoded nuclear proteins with impaired ability to induce apoptosis compared to wild-type pRb in vitro. Notably, three out of four tumors harboring RB1 mutations displayed primary resistance to treatment with either 5-FU/mitomycin or doxorubicin while only 14 out of 64 tumors without mutations were resistant (p = 0.046).

Conclusions: Although rare, our findings suggest RB1 mutations to be of pathological importance potentially affecting sensitivity to mitomycin/anthracycline treatment in breast cancer.

Show MeSH

Related in: MedlinePlus

Multiple sequence alignment of the pRb spacer region. A multiple sequence alignment of the spacer region between the A and B boxes of the pRb pocket was generated with ClustalX using default parameters [24]. The locations of two of the mutations reported here are marked with black arrows, and two previously reported mutations are shown in grey [32,33]. Sequences from eight different species were included in the alignment: Human [UNIPROT: P06400 RB_HUMAN], cow [UNIPROT: Q08E68_BOVIN], mouse [UNIPROT: P13405 RB_MOUSE], chicken [UNIPROT: Q90600 RB_CHICK], newt [UNIPROT: Q98966_NOTVI], salmon [UNIPROT: C0H9R0_SALSA], killifish [UNIPROT: Q5J3Q9_FUNHE], and zebrafish [UNIPROT: A0JMQ4_DANRE].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2908580&req=5

Figure 3: Multiple sequence alignment of the pRb spacer region. A multiple sequence alignment of the spacer region between the A and B boxes of the pRb pocket was generated with ClustalX using default parameters [24]. The locations of two of the mutations reported here are marked with black arrows, and two previously reported mutations are shown in grey [32,33]. Sequences from eight different species were included in the alignment: Human [UNIPROT: P06400 RB_HUMAN], cow [UNIPROT: Q08E68_BOVIN], mouse [UNIPROT: P13405 RB_MOUSE], chicken [UNIPROT: Q90600 RB_CHICK], newt [UNIPROT: Q98966_NOTVI], salmon [UNIPROT: C0H9R0_SALSA], killifish [UNIPROT: Q5J3Q9_FUNHE], and zebrafish [UNIPROT: A0JMQ4_DANRE].

Mentions: The two point mutations Leu607Ile and Arg621Cys are both located in the spacer region, previously assumed to be non-essential to pRb protein function [23]. Employing ClustalX using default parameters [24], a multiple sequence alignment of the pRb spacer region including sequences from eight different species was constructed. As shown in Figure 3, the spacer region is fairly well conserved. In fact, the human and mouse RB1-spacer sequences have a higher level of identity than the average human-mouse sequence identity (82% versus 70%). This finding indicates that the spacer region is of important for pRb function.


Identification and characterization of retinoblastoma gene mutations disturbing apoptosis in human breast cancers.

Berge EO, Knappskog S, Geisler S, Staalesen V, Pacal M, Børresen-Dale AL, Puntervoll P, Lillehaug JR, Lønning PE - Mol. Cancer (2010)

Multiple sequence alignment of the pRb spacer region. A multiple sequence alignment of the spacer region between the A and B boxes of the pRb pocket was generated with ClustalX using default parameters [24]. The locations of two of the mutations reported here are marked with black arrows, and two previously reported mutations are shown in grey [32,33]. Sequences from eight different species were included in the alignment: Human [UNIPROT: P06400 RB_HUMAN], cow [UNIPROT: Q08E68_BOVIN], mouse [UNIPROT: P13405 RB_MOUSE], chicken [UNIPROT: Q90600 RB_CHICK], newt [UNIPROT: Q98966_NOTVI], salmon [UNIPROT: C0H9R0_SALSA], killifish [UNIPROT: Q5J3Q9_FUNHE], and zebrafish [UNIPROT: A0JMQ4_DANRE].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2908580&req=5

Figure 3: Multiple sequence alignment of the pRb spacer region. A multiple sequence alignment of the spacer region between the A and B boxes of the pRb pocket was generated with ClustalX using default parameters [24]. The locations of two of the mutations reported here are marked with black arrows, and two previously reported mutations are shown in grey [32,33]. Sequences from eight different species were included in the alignment: Human [UNIPROT: P06400 RB_HUMAN], cow [UNIPROT: Q08E68_BOVIN], mouse [UNIPROT: P13405 RB_MOUSE], chicken [UNIPROT: Q90600 RB_CHICK], newt [UNIPROT: Q98966_NOTVI], salmon [UNIPROT: C0H9R0_SALSA], killifish [UNIPROT: Q5J3Q9_FUNHE], and zebrafish [UNIPROT: A0JMQ4_DANRE].
Mentions: The two point mutations Leu607Ile and Arg621Cys are both located in the spacer region, previously assumed to be non-essential to pRb protein function [23]. Employing ClustalX using default parameters [24], a multiple sequence alignment of the pRb spacer region including sequences from eight different species was constructed. As shown in Figure 3, the spacer region is fairly well conserved. In fact, the human and mouse RB1-spacer sequences have a higher level of identity than the average human-mouse sequence identity (82% versus 70%). This finding indicates that the spacer region is of important for pRb function.

Bottom Line: Multiple sequence alignment across species indicates the spacer to be evolutionary conserved.All three RB1 point mutations encoded nuclear proteins with impaired ability to induce apoptosis compared to wild-type pRb in vitro.Although rare, our findings suggest RB1 mutations to be of pathological importance potentially affecting sensitivity to mitomycin/anthracycline treatment in breast cancer.

View Article: PubMed Central - HTML - PubMed

Affiliation: Section of Oncology, Institute of Medicine, University of Bergen, Norway.

ABSTRACT

Background: The tumor suppressor pRb plays a key role regulating cell cycle arrest, and disturbances in the RB1 gene have been reported in different cancer forms. However, the literature reports contradictory findings with respect to a pro--versus anti--apoptotic role of pRb, and the consequence of alterations in RB1 to chemotherapy sensitivity remains unclear. This study is part of a project investigating alterations in pivotal genes as predictive factors to chemotherapy sensitivity in breast cancer.

Results: Analyzing 73 locally advanced (stage III) breast cancers, we identified two somatic and one germline single nucleotide changes, each leading to amino acid substitution in the pRb protein (Leu607Ile, Arg698Trp, and Arg621Cys, respectively). This is the first study reporting point mutations affecting RB1 in breast cancer tissue. In addition, MLPA analysis revealed two large multiexon deletions (exons 13 to 27 and exons 21 to 23) with the exons 21-23 deletion occurring in the tumor also harboring the Leu607Ile mutation. Interestingly, Leu607Ile and Arg621Cys point mutations both localize to the spacer region of the pRb protein, a region previously shown to harbor somatic and germline mutations. Multiple sequence alignment across species indicates the spacer to be evolutionary conserved. All three RB1 point mutations encoded nuclear proteins with impaired ability to induce apoptosis compared to wild-type pRb in vitro. Notably, three out of four tumors harboring RB1 mutations displayed primary resistance to treatment with either 5-FU/mitomycin or doxorubicin while only 14 out of 64 tumors without mutations were resistant (p = 0.046).

Conclusions: Although rare, our findings suggest RB1 mutations to be of pathological importance potentially affecting sensitivity to mitomycin/anthracycline treatment in breast cancer.

Show MeSH
Related in: MedlinePlus