Mutational analysis of CDKN2A genes in patients with squamous cell carcinoma of the skin

BACKGROUND: Nonmelanoma skin cancers [squamous cell carcinomas (SCC) and basal cell carcinomas (BCC)] are the most common neoplasias of the Caucasian population. OBJECTIVES: The purpose of our study was to determine the involvement of CDKN2A genes in the development of sporadic nonmelanoma skin cancer in Greek patients. PATIENTS AND METHODS: Allelic imbalance analysis was performed in 22 SCC and five Bowen's disease specimens. Mutational analysis was performed on exons 1alpha, 1beta and 2 of the CDKN2A locus in 22 SCC, five Bowen's disease and 39 BCC specimens. Exon 1alpha was additionally screened in 28 BCC specimens to complete the mutational analysis of a previous study. RESULTS: Overall, 52% (14 of 27) of the SCC and Bowen's disease specimens exhibited loss of heterozygosity (LOH) in at least one microsatellite marker, whereas, only two of 27 (7%) exhibited microsatellite instability. LOH in 9p appears to be equally involved in both BCC and SCC tumours. Exons 1alpha, 1beta and 2 of the CDKN2A locus were screened for mutations. A Val28Gly substitution in exon 1alpha and a CCC-->TTT (Ala57Val and Arg58Ter) substitution in exon 2, resulting in a change in the amino acid sequence, are reported for the first time in two SCCs, the latter being indicative of a combination of an ultraviolet (UV) radiation-induced mutation and a point mutation. A previously described polymorphism of CDKN2A, the gene for p16INK4a, Ala148Thr, was also detected in an allelic frequency of 3.72%. No mutation was found in any of the five Bowen's disease specimens, or in exon 1beta of CDKN2A, also the gene for p14ARF. CONCLUSIONS: Mutations and the high incidence of 9p LOH detected in our SCC samples imply that inactivation of CDKN2A genes, via allelic loss and/or mutation (probably UV-induced) may play a significant role in nonmelanoma skin cancer development, particularly in the more aggressive SCC type.     

Codon 12 Harvey-ras mutations are rare events in non-melanoma human skin cancer

ras mutations have been reported as an early event in some human malignancies and in the mouse skin model of multistep carcinogenesis; early studies in human non-melanoma skin cancers have reported variable rates of ras mutations. A recent study, however, has reported a high frequency of activating mutations of the Harvey- ras proto-oncogene in non-melanoma skin cancers, and the site specificity of the mutation at the second position of codon 12 prompted us to re-examine the importance of Ha- ras codon 12 mutations as an early event in the development of these tumours, using a combination of PCR and restriction fragment polymorphism of codon 12 of the Ha- ras gene. Dilution experiments confirmed that the method was sensitive and capable of detecting mutations at this codon when only 4% of the total alleles are mutated. We were surprised to find no mutations in the 40 basal cell carcinomas. 12 squamous cell carcinomas and 12 cases of Bowen's disease studied. We conclude that Ha- ras codon 12 mutations are rare events in human non-melanoma skin cancer in the U.K. The marked differences in the frequency of codon 12 Ha- ras mutations in published studies may relate to either technical artefacts, or differences in the molecular epidemiology between areas of low and high sun exposure.     

H—ras,N—myc及nm^23—H1癌基因蛋白在基底细胞癌中的表达

应用免疫组化技术研究１８例基底细胞癌，４例鳞状细胞癌，３例角化棘皮瘤及４例正常皮肤组织中Ｈ－ｒａｓ，Ｎ－ｍｙｃ及ｎｍ＾２３－Ｈ１癌基因蛋白的表达。结果表明：Ｈ－ｒａｓ蛋白产物主要分布于分化较成熟的组织，而Ｎ－ｍｙｃ则分布于持续生长的组织及较成熟组织，ｎｍ＾３－Ｈ１蛋白产物在基底细胞癌中表达最强，而在鳞状细胞癌中表达最低。     

Analysis of N- and K-ras mutations in the distinctive tumor progression phases of melanoma

Mutations in the ras genes are key events in the process of carcinogenesis; in particular, point mutations in codon 61 of exon 2 of the N-ras gene occur frequently in cutaneous melanoma. To investigate whether these mutations occur in early or late tumor progression phases, we searched for point mutations in the N- and K-ras genes in 69 primary cutaneous melanoma, 35 metastases, and seven nevocellular nevi in association with cutaneous melanoma. Lesions were microdissected in order to procure pure tumor samples from the distinctive growth phases of the cutaneous melanoma; the very sensitive denaturing gradient gel electrophoresis technique was used to visualize the mutations, and was followed by sequencing. Point mutations in the N-ras gene but not in the K-ras gene were detected on denaturing gradient gel electrophoresis. Twenty-three primary (33%) and nine metastatic (26%) melanomas showed bandshifts for N-ras. In the majority of cases, mutations occurring in early growth phases (i.e., the "intraepidermal" radial growth phase), were preserved in later growth phases (i.e., the invasive radial growth phase, vertical growth phase, and metastatic phase), which proves the clonal relationship between the successive growth phases. In three cases, however, the mutations differed between the distinctive growth phases within the same cutaneous melanoma, due to the occurrence of an additional mutation (especially in codon 61) in a later tumor progression phase. Our approach also permitted us to analyze the mutational status of nevi, associated with cutaneous melanoma. Six out of seven associated nevi carried the same sequence (mutated or wild-type) as the primary cutaneous melanoma, whereas in one case the sequence for N-ras differed between the primary melanoma and the associated nevus. In conclusion, this approach allowed us to demonstrate the clonal relationship between subsequent growth phases of melanoma and associated nevi; our results suggest that N-ras exon 1 mutations preferentially occur during early stages of tumor progression and hence may be involved in melanoma initiation, whereas those in N-ras exon 2 are found preferentially during later stages and hence are more probably involved in metastatic spread of cutaneous melanoma.     

UV-specific p53 and PTCH mutations in sporadic basal cell carcinoma of sun-exposed skin

UVB irradiation is known to produce DNA damage at mutation hotspots in the p53 tumor suppressor gene, leading to the development of skin cancers. Mutations in the PTCH tumor suppressor gene, which is known to be responsible for the development of nevoid basal cell carcinoma syndrome, have also been identified in sporadic basal cell carcinomas (BCCs). We describe the case of an 80-year-old welder in whom 3 novel p53 mutations, as well as UV-specific PTCH mutations, were detected in two BCC samples from sun-exposed skin. The simultaneous presence of UV-specific p53 and PTCH mutations in the same BCC sample has not previously been reported.     

Photocarcinogenesis: UVA vs. UVB radiation

Recent research is revealing combinations of disturbed oncogenic and tumor-suppressive signaling pathways by altered or missing genes in skin cancers: mutated PTCH (in the mitogenic Sonic Hedgehog pathway) and mutated p53 tumor suppressor gene in basal cell carcinomas (BCC), possibly an activated mitogenic RAS pathway and mutated p53 in squamous cell carcinomas (SCC), and possibly an activated MET/RAS pathway and inactive p16(INK4a) tumor suppressor in cutaneous melanomas. UV radiation damages DNA and can give rise to genomic alterations, varying from point mutations to crude chromosomal dislocations. UVB radiation (wavelength band 280-315 nm) is more carcinogenic than UVA radiation (315-400 nm) in experimental induction of SCC. The impact of UVB radiation can be clearly inferred from the characteristic point mutations in p53 found in human SCC and BCC. In contrast to UVB radiation, much of the mutagenic and carcinogenic action of UVA radiation appears to be mediated through reactive oxygen species (ROS). Experiments have shown that UVA1 (340-400 nm) exposure induces SCC largely without the characteristic point mutations in p53. Both UVB and UVA radiation can give rise to ROS-related point mutations (e.g. G to T) and crude genomic alterations (e.g. deletions) which may not be recognized as caused by UV radiation.     

Viral DNA detection and RAS mutations in actinic keratosis and nonmelanoma skin cancers

Background Actinic keratosis (AK) is a well‐established precancerous skin lesion that has the potential to progress to squamous cell carcinoma (SCC). Basal cell carcinoma (BCC) is a locally aggressive slowly growing tumour that rarely metastasizes. A number of viruses have been proposed to play a role in the development of nonmelanoma skin cancers (NMSC), but the most plausible evidence to date suggests that cutaneous human papillomavirus (HPV) is the key instigating factor. Objectives To evaluate the prevalence of HPV, cytomegalovirus (CMV), herpes simplex virus (HSV) and Epstein–Barr virus (EBV) and investigate their relationship with the presence of RAS gene mutations in cutaneous lesions obtained from nonimmunosuppressed patients. Methods HPV, CMV, HSV and EBV detection was performed using polymerase chain reaction (PCR) in skin biopsies (26 AK, 12 SCC and 15 BCC samples) that were collected from immunocompetent patients. The RAS mutation incidence was also investigated in all cutaneous lesions by use of PCR/restriction fragment length polymorphism and direct DNA sequencing. Results Seventeen out of 53 (32%) skin lesions were found to be positive for HPV DNA. The highest incidences of HPV infection were five of 15 (33%) in BCC and four of 12 (33%) in SCC specimens. The HPV incidence was eight of 26 (31%) in AK and eight of 53 (15%) in normal skin tissue. Twelve out of 53 (23%) skin lesions were CMV‐positive. The highest incidence of CMV infection was six of 15 (40%), observed in BCC specimens. The CMV incidence was two of 26 (8%) in AK and four of 12 (33%) in SCC. No normal skin biopsy was found to be positive for CMV. All cutaneous samples were negative for HSV and EBV DNA, as assessed by our PCR‐based assays. Only three samples, one AK (4%), one BCC (6%) and one SCC (8%), were found to carry a G>T transversion at the second position of HRAS codon 12. Both HRAS mutant SCC and BCC biopsies were HPV‐ and CMV‐positive, as well. Conclusions HPV DNA is detected in NMSC, AK and normal skin biopsies. Our results also indicate that CMV is involved in NMSC at higher levels than in premalignant lesions, whereas the virus was not detected in normal skin biopsies. HSV and EBV do not appear to be involved in the pathogenesis of cutaneous lesions. Moreover, we suggest that the HRAS codon 12 mutation is not a very common event in AK or NMSC. Finally, both viral infection and HRAS activation appear to represent independent factors in the aetiology of NMSC, samples of which were obtained from immunocompetent patients.     

Mutation spectra of epidermal p53 clones adjacent to basal cell carcinoma and squamous cell carcinoma

Foci of normal keratinocytes overexpressing p53 protein are frequently found in normal human skin. Such epidermal p53 clones are common in chronically sun-exposed skin and have been suggested to play a role in skin cancer development. In the present study, we have analyzed the prevalence of p53 mutations in epidermal p53 clones from normal skin surrounding basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Using laser-assisted microdissection, 37 epidermal p53 clones adjacent to BCC (21) and SCC (16) were collected. Genetic analysis was performed using a multiplex/nested polymerase chain reaction followed by direct DNA sequencing of p53 exons 2-11. In total, 21 of 37 analyzed p53 clones consisted of p53-mutated keratinocytes. The identified mutations were located in p53 exons 4-8, corresponding to the sequence-specific DNA-binding domain. All mutations were missense, and 78% displayed a typical ultraviolet signature. The frequency of p53 mutations was similar in skin adjacent to BCC compared to SCC. The presented data confirm and extend previous knowledge on the genetic background of epidermal p53 clones. The mutation spectra found in epidermal p53 clones resemble that of non-melanoma skin cancer. Approximately, 40% of the epidermal p53 clones lacked an underlying p53 mutation, suggesting that other genetic events in genes up- or downstream of the p53 gene can generate foci of normal keratinocytes overexpressing p53 protein.     

Harvey-ras gene expression and epidermal cell proliferation in dibenzo[a,l]pyrene-treated early preneoplastic SENCAR mouse skin

Topical application of dibenzo[a,l]pyrene (DB[a,l]P) to the dorsal skin of SENCAR mice induces codon 61 (CAA Gln to CTA Leu) mutations in the Harvey (H)-ras gene within 12 h after treatment. Between days 1 and 3, the frequency of these mutations increases rapidly, suggesting that skin cells carrying the codon 61 mutations proliferate in this period. We have investigated DB[a,l]P-treated mouse skin (12 h-7 d) for further evidence of H-ras expression and epidermal cell proliferation. Two waves of cell proliferation were observed: the first wave (1-2 d) correlated with the clonal proliferation of codon 61-mutated cells, and the second wave (3-7 d) correlated with DB[a,l]P-induced hyperplasia. DB[a,l]P-induced early preneoplastic cell proliferation correlated with H-ras and specific G1 cyclin expression. Total H-ras protein and cyclin D1 were found to increase during DB[a,l]P-induced hyperplasia, but the levels of guanosine triphosphate-bound (active) H-ras protein and cyclin E were increased during the putative clonal proliferation of codon 61-mutated cells. These results suggest that DB[a,l]P-induced oncogenically mutated cells proliferate in early preneoplastic skin. As this proliferation occurs in the absence of any promoting treatment, we propose that this phenomenon is a tumor initiation event.     

Allelic loss at Drosophila patched gene is highly prevalent in Basal and squamous cell carcinomas of the skin

The human homolog of the Drosophila Patched gene (PTCH), located at chromosome 9q22.3, is frequently altered in both nevoid basal cell carcinoma syndrome, and sporadic basal cell carcinomas (BCCs). However, alteration of the PTCH gene locus has been poorly studied in squamous cell carcinoma (SCC). We analyzed loss of heterozygosity (LOH) at five markers in and around the PTCH gene in 276 keratinocyte tumors from a population-based study in New Hampshire. We found a high prevalence of any 9q22.3 LOH in both BCC (75.5%) and SCC (60.8%), with BCC being significantly more likely to have LOH than SCC (P<0.009). The PTCH gene was specifically lost in 60% of BCC, and 50% of SCC tumors. Among SCC tumors, 9q22 LOH was significantly more likely to occur in those who tend to burn (P<0.05), and this association was strongest for tumors that occurred on sun-exposed areas of the body (P<0.04). Additionally, 9q22 LOH occurred more frequently in SCC tumors associated with a history of severe sunburns (P<0.08). Thus, in our large, population-based sample, 9q22 loss, including PTCH, was highly prevalent in both BCC and SCC. Overall, these data support the hypothesis that PTCH loss is a common, early lesion for SCC and BCC.     

皮肤鳞状细胞癌和基底细胞癌中p53和C-myc突变基因及蛋白的检测

目的 研究和探讨ｐ５３和Ｃ－ｍｙｃ基因突变在皮肤鳞状细胞癌（ＳＣＣ）和基底细胞癌（ＢＣＣ）的发生,发展中的作用。方法 采用多聚酶链反应－单链构象多态性分析（ＰＣＲ－ＳＳＣＰ）技术检测石蜡标本中ｐ５３与Ｃ－ｍｙｃ基因突变及免疫组化方法检测Ｃ－ｍｙｃ蛋白及突变型ｐ５３基因产物的表达。结果 ３０例ＳＣＣ中１２例ｐ５３基因突变（４０％）和１例Ｃ－ｍｙｃ基因突变,Ｐ５３蛋白表达占５０％（１５／３０）,Ｃ－ｍ     

Epigenetic alterations in sporadic basal cell carcinomas

Basal cell carcinoma (BCC) is the most common malignant human neoplasm characterized by slow growth and virtual absence of metastases. Recently, it has become evident that along with genetic mutations epigenetic alterations play a key role in the pathogenesis of human cancer. We searched for promoter methylation of hMLH1, RASSF1A, DAPK, APC, DCR1 and DCR2 genes and BRAF mutations in BCCs in association with the clinicopathological parameters and the histological subtypes of the tumours. Fifty-two BCCs, 17 FFPE along with 35 fresh tissue samples with matching normal tissues for 26 cases were analyzed by methylation-specific PCR to assess the methylation status of hMLH1, RASSF1A, DAPK, APC, DCR1 and DCR2 genes after sodium bisulfite treatment of the tumour and normal DNA. hMLH1 and DCR1 gene expression was investigated by immunohistochemistry. BRAF mutations were studied by high resolution melting analysis. Methylation was detected at a variable frequency of 44, 33, 32.5, 32 and 14 % of DCR2, APC, DCR1, RASSF1 and DAPK promoters, respectively, whereas methylation of hMLH1 promoter was absent. No BRAF mutations were found. There was no correlation between the frequency of the promoter methylation of the above-mentioned genes and the clinicopathological features or the histological subtypes of the tumours. The relatively high frequency of RASSF1A, DCR1, DCR2 and APC promoter methylation may imply that methylation constitutes an important pathway in the tumourigenesis of BCC that could provide new opportunities in developing epigenetic therapies for BCC patients. Nevertheless, further studies are needed to establish the above-mentioned hypothesis.     

Induction of skin papillomas, carcinomas, and sarcomas in mice in which the connexin 43 gene is heterologously deleted

It has been suggested that blocked gap junctional intercellular communication plays a crucial part in multistage carcinogenesis. The mouse skin tumor-promoting phorbol esters are potent inhibitors of gap junctional intercellular communication and this inhibition is considered to be a mechanism by which clonal expansion of "initiated" cells is promoted. We examined whether mice in which the gene for a gap junction protein, connexin 43, is heterozygously deleted are more susceptible to chemical carcinogenesis; connexin 43 is expressed in the basal cell layer and the dermis of the skin. When the back skin was painted with 7,12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol 13-acetate, the incidence and yields of both papillomas and carcinomas were similar in connexin 43+/- and connexin 43+/+ mice; for this experiment, the original mice with C57BL/6 genetic background was crossed with CD1 strain for three generations. Subcutaneous injection of 7, 12-dimethylbenz[a]anthracene resulted in induction of fibrosarcomas in connexin 43+/- and connexin 43+/+ mice to a similar extent. All papillomas and carcinomas induced with 7, 12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol 13-acetate contained the 7,12-dimethylbenz[a] anthracene-specific mutation in the ras gene (A to T transversion at the 61st codon). About 50% of fibrosarcomas also contained this mutation, but in the Ki-ras gene; there was no difference in the prevalence of this mutation in tumors from connexin 43+/- and connexin 43+/+ mice. None of the tumors examined, however, showed any mutation in the connexin 43 gene. These results suggest that the deletion of one allele of the connexin 43 gene does not significantly contribute to, nor alter, the molecular events involved in skin carcinogenesis. These results are compatible with previous observations that nongenetic disruption of function rather than mutations of connexins, commonly occurs in cancer cells.     

Urokinase plasminogen activator is immunocytochemically detectable in squamous cell but not basal cell carcinomas.

The presence of plasminogen activators (PA) in a variety of solid tumors appears to correlate, in a number of instances, with enhanced invasive or metastatic capabilities. In the present study, we have immunocytochemically examined basal cell (BCC) and squamous cell carcinomas (SCC) comprising a spectrum of histologic subtypes for the presence of urokinase-type (uPA) and tissue-type (tPA) PA. Neither uPA nor tPA was noted in any BCC, whether of the nodular, infiltrative, morpheaform, or basosquamous variety. uPA but not tPA was seen in 12 of 16 SCC examined; the tumors lacking uPA were all histologically well differentiated. No relationship between uPA expression and depth of invasion was noted, and uPA was not preferentially expressed at tumor borders. We conclude that uPA presence in SCC may relate to the degree of differentiation.     

Somatic mutations in the PTCH, SMOH, SUFUH and TP53 genes in sporadic basal cell carcinomas

BACKGROUND: Basal cell carcinoma (BCC) of the skin is the most common human cancer. The genetic alterations underlying BCC development are only partly understood. OBJECTIVES: To investigate further the molecular genetics of sporadic BCCs, we performed mutation analyses of 10 skin cancer-associated genes in 42 tumours. METHODS: Single-strand conformational polymorphism analysis followed by DNA sequencing was used to screen for mutations in the sonic hedgehog pathway genes PTCH, SMOH, SUFUH and GLI1, in the TP53 tumour suppressor gene, and in the proto-oncogenes NRAS, KRAS, HRAS, BRAF and CTNNB1. Microsatellite markers flanking the PTCH, SUFUH and TP53 loci at 9q22, 10q24 and 17p13, respectively, were studied for loss of heterozygosity (LOH). RESULTS: PTCH mutations were found in 28 of 42 tumours (67%). Microsatellite analysis revealed LOH on 9q22 in 20 of 38 tumours investigated (53%), including 14 tumours with and six tumours without PTCH mutations. SMOH mutations were identified in four of the 42 BCCs (10%) while two tumours demonstrated mutations in SUFUH, including one missense mutation and one silent mutation. None of the BCCs showed LOH at markers flanking the SUFUH locus. Seventeen BCCs (40%) carried TP53 mutations, with only three tumours showing evidence of biallelic TP53 inactivation. TP53 mutations were present in BCCs with and without mutations in PTCH, SMOH or SUFUH. Interestingly, 72% of the TP53 alterations were presumably ultraviolet (UV)-induced transition mutations. In contrast, only 40% of the PTCH and SMOH alterations corresponded to UV signature mutations. No mutations were identified in GLI1, NRAS, KRAS, HRAS, BRAF or CTNNB1. CONCLUSIONS: Our data confirm the importance of PTCH, SMOH and TP53 mutations in the pathogenesis of sporadic BCCs. SUFUH alterations are restricted to individual cases while the other investigated genes do not appear to be important targets for mutations in BCCs.     

Molecular genetic analysis of the BRCA2 tumor suppressor gene region in cutaneous squamous cell carcinomas

Background:  Germ line mutations of the BRCA2 tumor suppressor gene with subsequent loss of the remaining wild-type BRCA2 allele have been identified in up to 35% of familial breast cancer cases. A high frequency of allelic loss at the BRCA2 gene locus has also been reported in a variety of sporadic epithelial tumors including oesophageal squamous cell carcinomas (SCC), and sporadic head and neck SCC.
Aim:  The present study aimed to examine the integrity of the BRCA2 gene in cutaneous SCC.
Materials and methods:  Allelic imbalance/loss of heterozygosity (AI/LOH) was examined in 22 histologically confirmed cutaneous SCC at two microsatellite markers, D13S260 (centromeric to the BRCA2 gene) and D13S267 (telomeric to the BRCA2 gene). Immunohistochemical analysis of BRCA2 protein expression was also examined in the cutaneous SCC.
Results:  AI/LOH at the D13S260 locus was found in eight of the 19 informative SCC, and AI/LOH at the D13S267 locus was found in 12 of the 18 informative SCC. Seven SCC showed allelic loss at both markers, and six SCC showed retention of heterozygosity at both markers. Expression of BRCA2 protein was only detected in six of the normal epidermises and three of the 21 SCC examined.
Conclusion:  AI/LOH of the BRCA2 gene region was found to be common in the cutaneous SCC.     

二例可变性红斑角化症患者GJB3、4基因突变研究

目的 探讨2例散发可变性红斑角化症（EKV）患者的GJB3和GJB4基因突变。 方法 提取EKV患者、家族成员及正常人基因组DNA,采用PCR扩增GJB3和GJB4基因所有外显子及其邻近的剪切点,进行双向直接测序。结果 1例EKV患者GJB4基因未见变化,GJB3基因的第134位碱基鸟嘌呤（G）被胞嘧啶（C）替换,导致蛋白质第45位的甘氨酸转换成丙氨酸（G45A）。另1例EKV患者GJB3、GJB4均未发现突变。结论 1例 EKV患者存在GJB3基因G45A错义突变。     

Reduction of expression of tuberin,the tuberous-sclerosis-complex-gene-2 product in tuberous sclerosis complex associated connective tissue nevi and sporadic squamous and basal cell carcinomas

BACKGROUND: Patients affected with tuberous sclerosis complex (TSC) are prone to the development of multiple benign tumors of the skin and other organs. Tuberin, the protein product of the tuberous-sclerosis-complex-2 tumor suppressor gene (TSC2) has been shown to inhibit cell proliferation. In TSC associated kidney tumors and sporadic brain tumors the loss/reduction of tuberin has been shown. METHODS: Specimens of nine squamous cell carcinomas (SCC) and five basal cell carcinomas (BCC) from patients without TSC and six biopsies of connective tissue nevi (CTN) of patients with TSC were obtained. Specimens were analyzed by immunoblotting for the expression of tuberin. RESULTS: Absent or reduced levels of tuberin were detected in the dermal parts of three of six shagreen patches, two of five BCC, and four of nine SCC. CONCLUSIONS: In tumors/hamartomas of patients with TSC the complete loss of TSC2 and tuberin is a mechanism which could be shown for CTN, thereby excluding the possibility of haploinsufficiency of TSC2. In a substantial number of cutaneous BCC and SCC the loss or downregulation of tuberin seems to be epigenetic, as alterations of TSC2 are not known in these tumors. The absence or reduction of tuberin might contribute to their proliferation.